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First published online 7 February 2007
doi: 10.1242/dev.02817

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Redundancy and specialization among plant microRNAs: role of the MIR164 family in developmental robustness

¹ Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
² Institute of Plant Biology, University of Zurich, Zollikerstrasse 107, 8008 Zurich, Switzerland.

View larger version (31K): [in this window] [in a new window]   Fig. 1. Floral phenotype of mutant plants impaired in miR164 biogenesis. (A) Quantification of miR164 abundance in mir164 mutants. RNA blot analysis of the small RNA fraction isolated from wild-type Col-0, mir164a-4, mir164b-1, mir164c-1, mir164c-2, and mir164a-4 b-1 c-1 triple-mutant inflorescences hybridized with probes complementary to miR164a (upper blot) and U6 small RNA (middle blot), respectively. The ethidium bromide-stained agarose gel is shown beneath (numbers indicate fold-change of miR164 accumulation with respect to Col-0 wt, which was set to 1). As miR164a and miR164b differ from miR164c in a single nucleotide, different miR164 miRNAs cannot be distinguished on an RNA blot; thus, signals are derived from all three miR164 miRNAs. The experiment was repeated twice with the same result. The antisense MIR164 oligonucleotide probe hybridizes to two distinct RNA size classes, of 21 and 24 nt, in agreement with previous reports (Dunoyer et al., 2004; Valoczi et al., 2006). It has been proposed that the 21 nt form of miR164 is the functional entity sufficient to guide target cleavage, for which the 24 nt form, which has distinct requirements for its biogenesis, appears to be dispensable (Dunoyer et al., 2004). (B-D) Results of SEM analysis. (B) Mature (stage 13) wild-type flower of accession Ler. Flower stages were defined according to Smyth et al. (Smyth et al., 1990). (C) Stage 12 and (D) stage 13 flowers of mir164abc triple-mutant plants show variable organ numbers and unfused carpels. Sepals have been removed for better visibility of the inner organs. Scale bars: 200 µm in B; 100 µm in C,D. Abbreviations: pe, petals; ca, carpels; st, stamens. (E) A mir164abc triple-mutant inflorescence. (F,G) Charts representing organ counts from mir164abc triple-mutant (black) and mir164aAbBcC plants (gray), which served as the wild-type control to assess the potential influence of the mixed Ler/Col-0 background on the phenotypic changes. The average floral organ number ('Organ count') is plotted against each flower position along the stem ('Flower'). Numbers indicate the position of the flower along the stem from the oldest (1) to the youngest (25). Error bars represent s.d. in (F) sepal and (G) petal number. Stamen number was reduced with respect to Col-0 and slightly reduced with respect to the wild-type control. Notably, variability in stamen number, but not in sepal and petal number, increased in the mixed Ler/Col-0 background, when compared with the Col-0 background (data not shown). Carpel number is only weakly affected in mir164abc mutants.

View larger version (114K): [in this window] [in a new window]   Fig. 2. Phyllotaxis defects of mir164abc triple-mutant plants. (A,B,F-I) SEM pictures and (C-E) photographs of Col-0 wild-type plants (A,E,F,H) and mir164abc mutants (B,C,D,G,I). (A,B) Initiating flower primordia follow a spiral phyllotactic pattern in the wild type (A) and in mir164abc triple mutants (B). Numbers indicate the succession of floral bud initiation. (B) Arrowheads in B point to sepal primordia of different sizes. (C-E) Flowers of mir164abc triple-mutant plants are arranged randomly along the stem (C,D) when compared with the regular pattern of Col-0 wild-type flowers (E). i, internode. (F-I) Stem internodes are uniformly covered with long and rectangular epidermal cells in the wild type (F,H), whereas clustered flowers in mir164abc triple-mutant plants are separated by few, variably shaped non-elongate cells (G,I). In H,I, the margins of equivalent cells are highlighted to demonstrate the differences in cell shape and size. (J) Distribution of size classes, each comprising a specific internode length. The number of internodes ('Number/category') falling into a specific size category are plotted against the size categories ('Size category [mm]'). The internode sizes of the mixed Ler/Col-0 wild-type control (wt, gray) are distributed around the mean value 8.7±3.6 (s.d., ntot=149), whereas internode distribution of the mir164abc mutant (m, black) does not follow a similar pattern. (K) The average internode distance (in mm) is 8.7±3.6 (s.d., ntot=149) for the wild-type control (wt, gray) and 8.6±8.6 (s.d., ntot=150) for mir164abc triple-mutant (m, black) plants. Error bars indicate s.d. Scale bars: 20 µm in A,B; 100 µm in F-I.

View larger version (159K): [in this window] [in a new window]   Fig. 3. Expression patterns of miR164 miRNAs in wild-type plants. (A-D) Confocal images of inflorescences. Each transgenic plant expresses the GFP variant 3xVENUS-N7 (green/yellow) under control of the individual miRNA regulatory sequences (as indicated). In A,B, FM4-64 dye was used to stain plasma membranes (red); in C,D, organ outlines are highlighted by red chlorophyll autofluorescence. T1 plants were examined and representative expression patterns are shown. The number of plants showing depicted expression pattern (x) with respect to total sample size (ntot), indicated as ratio (x/ntot), was 7/7 (A), 2*/20 (B) and 5/5 (C,D). *No expression was detected in 18 out of 20 transgenic lines harboring pMIR164b::3xVENUS-N7. (E-P) In situ hybridization analysis of miR164 miRNA distribution (E,F,I-P) using DIG-labeled LNA-ath-miR164a antisense oligos, in Ler wild-type (E,F), in mir164a-4 b-1 double-mutant (I,J), in mir164a-4 c-1 double-mutant (K,L), in mir164b-1 c-1 double-mutant (M,N) and in mir164abc triple-mutant (O,P) plants. The inset in K shows mir164 accumulation in developing flowers. (G,H) No signal above background was detected when DIG-labeled Scramble-miR LNA-oligo was used as a control probe on Ler wild-type tissue. Scale bars: 100 µm.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Loss of miR164 miRNA-mediated regulation quantitatively affects target gene expression. (A-C) Bar charts showing the results of relative qRT-PCR experiments, in which the target transcript abundance was assessed for different tissue types (as indicated) of wild-type and mir164abc triple-mutant plants. Results were normalized using TUB4 transcript levels. The transcript abundance of all predicted miR164 targets is higher in the mir164abc triple mutant (m) as compared with the wild type (wt) in all tissues tested. Fold-change differences in transcript levels between the wild type and the mir164abc triple mutant are shown. Bars represent the s.e. of the measurements.

View larger version (107K): [in this window] [in a new window]   Fig. 5. Effect of miR164 miRNA regulation on target gene expression. Representative confocal images of inflorescences of primary transformants. FM4-64 dye was used to stain plasma membranes (red). (A-D) Effects of miR164-mediated regulation on CUC1 and CUC2 expression. Consequences of permitted (A,B) and abolished (C,D) miR164-mediated regulation for translational fusions of CUC1 (A,C) and CUC2 (B,D) to GFP. The same confocal microscopy settings have been used for the images shown. Arrowheads (A,B) mark cells that weakly express GFP in boundaries between the inflorescence meristem and flower primordia. (E,F) Transcriptional reporters for CUC1 (E) and CUC2 (F) expressing the GFP variant 3xVENUS-N7 (green). Number of plants showing depicted expression pattern (x) with respect to total sample size (ntot), indicated as ratio (x/ntot), was 7/9 for pCUC1::CUC1-GFP (A), 6/6 for pCUC1::CUC1m-GFP (C), 10*/20 (*no expression detected in others) for pCUC2::CUC2-GFP (B), 4/4 for pCUC2::CUC2m-GFP (D), 7/8 for pCUC1::3xVENUS-N7 (E) and 6/7 for pCUC2::3xVENUS-N7 (F). Scale bars: 100 µm.

View larger version (113K): [in this window] [in a new window]   Fig. 6. Target mRNA accumulation in mir164abc triple mutants. (A-F) In situ localization of CUC2 (A,B) and CUC1 (C-F) transcripts in transverse sections of stage 9 flowers (A,B) and of inflorescences (C-F). Tissue of wild-type (A,C,E) and mir164abc mutant (B,D,F) plants was processed equivalently and was present on the same microscope slide (A,B), or was hybridized in the same slide sandwich (C,D), to allow a direct comparison of the signals obtained. Arrows (A,B) point to regions of elevated CUC2 expression in partially fused carpels (ca) of mir164abc mutants as compared with the wild type. By contrast, CUC2 expression in stamens (st) appeared to be unaffected. (C,D and their enlargements E,F) Randomly located foci of high CUC1 expression were sometimes observed within primordia (asterisks) and between primordia (arrowheads) of mir164abc mutant plants. Scale bars: 20 µm.

View larger version (123K): [in this window] [in a new window]   Fig. 7. CUC1 acts as a growth antagonist. (A-C) SEM images of the (abaxial) sepal epidermis of wild-type Ler (A), mir164abc triple-mutant (B), and 35S::CUC1m-GFP transgenic plants (C). The sepals of mir164abc mutants were typically narrower than, but otherwise indistinguishable from, wild-type sepals (compare B with A). (D-G) The bar chart in D depicts the average number of epidermal sepal cells touching a 100 µm by 100 µm square projected onto the central abaxial region of sepals of wild-type (G) and 35S::CUC1m-GFP transgenic plants (F). The average cell number per 0.01mm2 was 31.8±8.8 (s.d., ntot=10) for 35S::CUC1m-GFP transgenic plants and 28.1±2.5 (s.d., ntot=11) for Ler wild-type sepals. The bar chart in E depicts the average sepal length of 35S::CUC1m-GFP and wild-type plants. The average sepal length was 360±101 µm (s.d., ntot=10) for 35S::CUC1m-GFP plants and 1529±89 µm (s.d., ntot=9) for Ler wild-type plants. Scale bars: 100 µm.