Article Figures & Data
Figures
- Figure 1.
Fold Back Structure, Expression, and Conservation of miR824 in the Brassicaceae.
(A) 5′ to 3′ stem-loop hairpin structures of the pre-miR824 predicted for Arabidopsis and three closely related Brassica species. The mature miR824 sequence is indicated in red, and the miR824* sequence is indicated in blue. G:U wobble pairing is shown by a circle. Note that the miR824/miR824* sequences are identical in Arabidopsis and the Brassica species.
(B) RNA blot hybridization of miR824 in leaves of Brassica species, Arabidopsis Col-0, O. sativa, and N. benthamiana. The 5S rRNA and tRNA loading standards are stained with ethidium bromide. The size of the RNA is indicated at left in nucleotides (nt).
(C) RNA ligase–mediated rapid amplification of cDNA ends (RLM-RACE) mapping of AGL16 mRNA cleavage sites in Col-0. The arrow indicates the site of cleavage in 10 of 10 sequenced clones. Perfect base pairing between miR824 and AGL16 mRNA is shown by vertical lines; G:U wobble pairing is indicated by a circle. The predicted free energy of pairing between miR824 and AGL16 mRNA is −38.6 kcal/mol. Conserved miRNA binding sites (red), conserved nucleotides (black), and nonconserved nucleotides (gray highlight) in Arabidopsis and Brassica species are indicated.
(D) to (F) Sequence complementarity between transcripts encoded by miRNA genes and their targets. The red line in (D) indicates complementarity between MIR824 and AGL16 in Arabidopsis. The orange line in (E) indicates complementarity between the MIR824 ortholog in B. oleracea (MIRBr824) and its target sequence AGL16. The blue lines in (F) indicate complementarity between MIR171 and its two targets genes, SCL6-III and SCL6-IV, in Arabidopsis. The gray line in each panel indicates the expected background complementarity at each position in the duplex (see Supplemental Methods online). The percentage of paired bases in a window of 20 nucleotides is shown for a potential duplex structure between miRNA precursor and target sequences. The duplex was anchored at the miRNA complementary site, indicated by vertical dashed lines. Values for each window are plotted aligned to its center position. Sequences of miRNA gene transcripts and their target RNAs, 88 nucleotides in length and centered on the miRNA complementary site, are shown below each plot with the miRNAs highlighted in red.
- Figure 2.
Expression of the MIR824 Locus in the Wild Type and in the Overexpression Mutant m3.
(A) Schematic representation of the MIR824 locus (At4g24415.1) on chromosome IV based on the alignment of ESTs with genomic sequences (see Supplemental Figure 2A online). Exons (open boxes), introns (horizontal lines), start of transcription (+1), transcript length, and the position and orientation of miR824 (short arrows) are indicated. The scheme is not drawn to scale.
(B) to (D) Transient expression experiments with pProMIR824:Luc constructs. Col-0 plants were bombarded with pPro2x35S:Luc carrying a luciferase gene regulated by the Pro2x35S promoter (B), pProMIR824:Luc carrying 2954 bp of genomic region upstream of the pri-miR824 start of transcription fused to the luciferase gene of pProMIR824:Luc (C), and pLuc carrying a promoterless luciferase/35S terminator cassette (D). Yellow to red false color indicates increasing levels of luciferase activity detected at 48 h after bombardment.
(E) RNA gel blot hybridization of miR824 in 20 μg of low molecular weight RNA and AGL16 mRNA in 2 μg of poly(A)+ RNA prepared from leaves of Col-0 and m3. tRNA and 5S rRNA were stained with ethidium bromide. The fold expression of RNA in m3 relative to Col-0 normalized for β-TUBULIN mRNA is indicated. The sizes of the RNAs are shown at left in nucleotides (nt).
- Figure 3.
Expression of miR824 and AGL16 mRNA in AGL16.1/2 and AGL16m1/2 Leaves.
(A) Nucleotide sequence of AGL16 (AGL16m) with silent mutations in the miR824 recognition site. miR824 pairs to AGL16 RNA at the nucleotide segment corresponding to amino acids Ser-197 and Leu-203. Predicted free energies of pairing to miR824 are indicated at left. Mutated nucleotides are underlined. Vertical lines indicate perfect base pairing, circles indicate G:U wobble pairing, and gray shading indicates the HincII restriction site (GTT/GAC) introduced into AGL16m.
(B) Fold expression relative to the wild type of uncleaved target AGL16 mRNA (black bars) and SCL6-III mRNA (gray bars), determined by RT–quantitative PCR using primers spanning the miR824 and the miR171 complementary sites, was normalized to TIP41-like (At4g34270) mRNA.
(C) RNA gel blot hybridization of low molecular weight RNA in leaves of Col-0, AGL16.1/2, and AGL16m1/2 using probes for miR824 and miR171. tRNA and 5S rRNA were stained with ethidium bromide. The sizes of the RNAs are indicated at left in nucleotides (nt).
(D) AGL16 and AGL16m transcripts were distinguished by HincII digestion, which only digests AGL16m cDNA, after RT-PCR amplification of endogenous and AGL16m transcripts.
- Figure 4.
Effects of Altered AGL16 Expression on the Proportion of Primary and Higher-Order Stomatal Complexes.
(A) Relative proportion of primary (black bars), secondary (white bars), tertiary (gray bars), and quaternary (striped bars) stomatal complexes on the abaxial surfaces of the fifth rosette leaves of Col-0, AGL16.1/2, AGL16m1/2, m3, and agl16-1 plants. At least 110 stomatal complexes were scored for each line. Asterisks above the bars indicate distributions significantly different (P < 5 × 10−5) from the Col-0 distribution by the χ2 test.
(B) and (C) Representative scanning electron microscopy images of stomatal complexes on the abaxial surfaces of fifth rosette leaves. Bars = 10 μm.
(B) A primary Col-0 stomatal complex consisting of a central pair of guard cells (G1 and G2) and stoma surround by neighboring cells (E1, E2, and E3).
(C) A quaternary stomatal complex of AGL16m1 with primary, secondary, tertiary, and quaternary complexes. The numbered arrows indicate the apparent order in which stomata form. Note that the fourth order stomata is at the early, satellite meristemoid stage of development.
- Figure 5.
Time Course of Stomatal Development on the Abaxial Surfaces of First Rosette Leaves.
Drawings of the relevant features of nail polish pictures taken from sequential dental resin impressions showing the development of representative stomatal complexes on the abaxial surfaces of first true leaves of Col-0 (A), AGL16.1 (B), agl16-1 (C), and AGL16m1 (D) plants monitored daily starting at 6 d after germination (dpg). Satellite meristemoids are colored in green, guard mother cells in yellow, kidney-shaped guard cells in gray, and jigsaw-shaped epidermal cells are not colored. Bar = 10 μm.
- Figure 6.
Effects of Altered AGL16 mRNA Expression on Stomatal Density and Stomatal Index.
(A) and (B) Average stomatal density (A) and average stomatal index (B) (SI%) ± sem for three to four replicates of the abaxial surfaces of fifth rosette leaves.
(C) Average densities of primary stomatal complexes and higher-order stomatal complexes calculated from the individual measurements in (A) and the proportion of stomatal types in Figure 4A. Significance levels (t test of means) relative to the Col-0 controls were as follows: * P < 0.05, ** P < 0.025, *** P < 0.01, and **** P < 0.005.
- Figure 7.
Expression Patterns of miR824 and AGL16 mRNA in Stomatal Complexes.
In situ hybridization of the abaxial epidermis of first true leaves of B. rapa imaged at 1 week after germination. Differential interference contrast images show whole mounts hybridized with probes for sense miR824 ([A] and [B]), antisense miR824 (C), sense AGL16 mRNA (D), and antisense AGL16 mRNA (E). Regions hybridizing with the probe appear dark gray. Mature stomata (S), satellite meristemoids (SM), and guard mother cell (GMC) are indicated. Bars = 10 μm.
Additional Files
Supplemental Data
Files in this Data Supplement:
- Supplemental Figure 1 - Expression of miR824 and AGL16 mRNA in Arabidopsis miRNA mutants and Brassica species.
- Supplemental Figure 2 - Genomic locations of MIR824 ESTs and the T-DNA insertion in line m3.
- Supplemental Figure - A partial characterization of the Arabidopsis AGL16 insertion mutant agl16-1.
- Supplemental Figure 4 - The phenotype of transgenic Arabidopsis plants altered in AGL16 mRNA expression.
- Supplemental Figure 5 - The positioning of stomatal complexes on the abaxial epidermis of transgenic Arabidopsis plants altered in AGL16 mRNA expression.
- Supplemental Table 1 - Sequences of oligonucleotides used in this study.
- Supplemental Methods - Construction of the pAGL16 and pAGL16m binary vectors and the Luciferase reporter gene vector pProMIR824:Luc and procedure used to study sequence complementarity between MIR824 and AGL16 transcripts.
