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First published online June 28, 2004
doi: 10.1242/10.1242/dev.01206

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Modulation of floral development by a gibberellin-regulated microRNA

¹ Department of Cell and Developmental Biology, John Innes Centre, Norwich NR4 7UH, UK
² Sainsbury Laboratory, John Innes Centre, Norwich NR4 7UH, UK

View larger version (29K): [in a new window]   Fig. 1. MiR159 and GAMYB mRNA target sequences are evolutionarily conserved. (A) Sequence similarity between three Arabidopsis miR159 isoforms. The nucleotide mismatches are in grey. (B) Sequence similarity between miR159a and internal sequences of putative target genes. Nucleotide mismatches between the internal sequences and miR159a are in grey, and the total number of mismatches is indicated after each sequence. Function and accession number for each putative target is indicated on the right. (C) Location of the complementary sequence between GAMYB genes and miR159. Regions of similarity between Arabidopsis and H. vulgare GAMYB genes are shown in black. The region of sequence that is complementary between miR159a and the GAMYB genes is shown in grey.

View larger version (69K): [in a new window]   Fig. 5. Overexpression of miR159a delays the floral transition in short days (SDs). (A) wild-type (WT) and 35S:miR159a homozygotes grown for 4 weeks in SDs. The arrow indicates a new rosette leaf. (B) WT and 35S:miR159a homozygotes grown for 6 weeks in short days. (C) Means of the number of total leaves produced before flowering (nb), and of the number of days to flower in SD conditions (±s.e.; n>10), are shown. Wild type, light grey bars; 35S:miR159a homozygotes, dark grey bars. (D) RNA gel-blot analysis of miR159 levels in 4-week-old wild-type and 35S:miR159a leaves. Lines 2 and 4 are clonally independent (T1) transgenic lines that segregate 35S:miR159a. WTF, segregants lacking the transgene, flowered at the same time as wild type (non-transgenic control); LF, late flowering segregants carrying 35S:miR159a. (E) RNA gel-blot analysis of MYB33, LEAFY and SOC1 mRNA performed on the same plant material as in D. ELF4a transcript was used as a RNA sample control.

View larger version (98K): [in a new window]   Fig. 6. 35S:miR159a plants have reduced fertility. (A) Wild type and 35S:miR159a homozygotes grown for 4 weeks in a LD photoperiod (16 hours light). The arrows indicate the two last cauline leaves formed. (B) Siliques of wild type and 35S:miR159a homozygotes. Scale bar: 3 mm. (C) Wild-type inflorescence. (D) Inflorescence of 35S:miR159a homozygote. (E) RNA gel-blot analysis of miR159 and MYB33 from wild-type and 35S:miR159a flowers.

View larger version (95K): [in a new window]   Fig. 7. Overexpression of miR159a affects anther development. (A,C) Wild-type flowers. (B,D) Flowers of 35S:miR159a homozygotes. (E) Anthers from wild-type and 35S:miR159a homozygote [taken from flowers at floral development stage 10 (Cheng et al., 2004)]. (F) Anthers from wild-type and 35S:miR159a homozygotes (from flowers at floral development stage 12). Scale bars: A, B, 1 mm; C, D, 500 µm; E, 50 µm; F, 100 µm.

View larger version (44K): [in a new window]   Fig. 2. MiR159 directs the cleavage of MYB33 transcripts and regulates MYB33 levels. (A) Constructs specifying miR159a, MYC-MYB33 and MYC-mMYB33 RNAs and driven by the 35S promoter. 35S:GFP was used as control for co-Agro-inoculation. (B) RNA gel-blot analysis of miR159, MYC-MYB33 and MYC-mMYB33 RNA forms (full length MYC-MYB33a and cleaved 3'-MYB33b transcripts) in N. benthamiana leaves co-Agro-inoculated with different combinations of 35S:miR159a, 35S:MYC-MYB33, 35S:MYC-mMYB33 and 35S:GFP as indicated. (C) Immunoblot analysis of total proteins (20 µg) from N. benthamiana leaves co-Agro-inoculated as in B. Goat anti-myc antiserum and a peroxidase-conjugated anti-goat IgG were used as primary and secondary antibodies, respectively. The lower panel shows a GFP immunoblot using the same extracts (loading control).

View larger version (29K): [in a new window]   Fig. 3. MiR159 levels vary in different tissues. RNA gel-blot analysis of miR159 from Arabidopsis root, 5 day-old seedling, rosette leaf, cauline leaf, flower and silique tissues; N. benthamiana leaf and flower tissues; barley (Hordeum vulgare) leaf, apical shoot and green spike (inflorescence) tissues. 5S rRNA was used as RNA control (shown in ethidium bromide-stained gel).

View larger version (36K): [in a new window]   Fig. 4. GA regulates miR159 levels via the DELLA-dependent GA signalling pathway. (A) RNA gel-blot analysis of miR159, MYB33 and SOC1 from wild-type (WT), gai, ga1-3 and ga1-3 gai-t6 rga-24 plants grown in long days (LD) in the presence (+GA) or absence (–GA) of gibberellin (100 µM GA3 sprayed twice per week). The blots were stripped and re-analysed with miR167 and ELF4a probes (RNA controls). (B) RNA gel-blot analysis of miR159 levels in wild-type and sln1-dwarf mutant barley (Hordeum vulgare). 5S rRNA was used as RNA control.

View larger version (10K): [in a new window]   Fig. 8. The GA-DELLA signalling system regulates floral development by modulation of miR159/GAMYB and SOC1. Schematic representation of the regulation of the floral transition in SD photoperiods via the GA-DELLA signalling pathway. GA relieves the DELLA repression of GAMYB (e.g. MYB33) and SOC1 and enhances activity of the downstream floral meristem identity gene LEAFY. This activation is moderated by the GA activation of miR159, a post-transcriptional regulator of GAMYB transcript levels. This GA-dependent homeostatic mechanism provides sensitive regulation of the floral transition in SDs and anther development via the regulation of GAMYB levels.