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First published online 16 August 2006
doi: 10.1242/dev.02521

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Temporal regulation of shoot development in Arabidopsis thaliana by miR156 and its target SPL3

Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018, USA.

View larger version (28K): [in a new window]   Fig. 1. SPL3, SPL4 and SPL5 are structurally similar and produce transcripts that are cleaved by miR156. (A) Genomic structure of SPL3, SPL4 and SPL5. White boxes indicate untranslated sequences; grey boxes indicate coding sequences; horizontal lines indicate introns; vertical lines indicate transcription start site or polyadenylation site identified by 5 ' or 3 ' RACE. (B) Cleavage sites identified by 5 'RLM-RACE.

View larger version (46K): [in a new window]   Fig. 2. SPL3 promotes vegetative phase change and floral induction and is repressed by miR156. (A) Structure of constructs used in this study. The sequence of the mutated miR156 target site is illustrated. A wild-type site is indicated by a black line and a mutated site is indicated by a grey line. (B) GUS expression (top) and RT-PCR of GUS mRNA (bottom) in progeny of crosses between transgenic plants constitutively expressing miR156a, and miR156-sensitive or insensitive versions of the GUS-SPL3 mRNA. (C) Morphology of transgenic plants expressing miR156-sensitive (SPL3) and miR156-insensitive (SPL3m, SPL3) versions of SPL3 under the regulation of the 35S promoter. (D) The number of leaves without abaxial trichomes (black), with abaxial trichomes (grey), cauline leaves (white) and flowering time (days after planting, top of bar) for the genotypes illustrated in C (± s.e.m.). Plants transformed with 35S::SPL3 are not significantly different from control plants. 35S::SPL3m and 35S::SPL3 have significantly fewer juvenile, adult and cauline leaves than control plants (n>30 for each genotype, P<0.01). (E) The effect of 35S::SPL3m on the morphology of leaves 1 and 2. This transgene produces a significant decrease in the length of the petiole and a slightly more acute leaf base (n=30 for each genotype; P<0.01).

View larger version (24K): [in a new window]   Fig. 3. miR156 promotes juvenile development. (A) Structure of the constructs used to overexpress miR156. DNA from the intergenic region containing miR156 was placed under 35S regulation. (B) Morphology of transgenic plants expressing the constructs illustrated in A. (C) The number of leaves without abaxial trichomes (black), with abaxial trichomes (grey) and flowering time (dap) in transgenic plants with an empty vector or 35S::miR156a. (D) The number of leaves without abaxial trichomes (black) and with abaxial trichomes (grey) in the F2 progeny of plants hemizygous for 35S::miR156a and 35S::SPL3m. (E) Successive rosette leaves from representative F2 plants illustrated in B. Only some of the leaves of 35S::miR156a are illustrated.

View larger version (35K): [in a new window]   Fig. 4. SPL4 and SPL5 promote vegetative phase change and flowering, and are repressed by miR156. (A) The number of leaves without abaxial trichomes (black), with abaxial trichomes (grey) and flowering time (dap, top of bar) in plants homozygous for T-DNA insertions in SPL3; SALK_035860 was isolated in Col, whereas FLAG_173C12 was isolated in Ws. The small delay in abaxial trichome production in FLAG_173C12 is significant (n=22 for each genotype; P<0.05). (B) The number of leaves without abaxial trichomes (black), with abaxial trichomes (grey), and flowering time (dap, top of bar) in plants constitutively expressing SPL4 and SPL5 transcripts with or without () a 3 ' UTR. Error bars=s.e.m. (C,D) Morphology of the transgenic lines illustrated in B.

View larger version (34K): [in a new window]   Fig. 5. miR156 is responsible for the temporal expression of SPL3. (A) Blots of total RNA from the shoot apex of plants grown for different lengths of time in SD. The approximate number of leaves greater than 100 µm in length present at each time point is indicated in parentheses. Actin was used as a loading control for SPL3, and U6 was used as a loading control for miR156. (B) GUS expression in transgenic plants of different ages. Leaf number 7 is indicated. The approximate number of leaves greater than 100 µm in length in these samples is indicated in parentheses. (C) Quantitative analysis of GUS activity in extracts of the lines illustrated in B. Error bars indicate s.e.m. (D) Northern analysis of miR156 levels in the transgenic lines illustrated in B and C.

View larger version (42K): [in a new window]   Fig. 6. zip-1 and rdr6-11 affect the amplitude of SPL3 expression, but not its temporal pattern. (A) Northern analysis of SPL3 and miR156 RNA in SD-grown plants at 14 dap (6 leaves) and 21 dap (12 leaves). zip-1 and rdr6-11 produce a modest increase in SPL3 mRNA without affecting the level of miR156. hst-6 reduces the accumulation of miR156 and produces a dramatic increase in SPL3 mRNA at 21 dap. U6 was used as a loading control for miR156 and 25S rRNA was the loading control for SPL3. (B) Northern analysis of SPL3 mRNA in SD-grown wild-type and mutant plants of different ages. Hybridization intensity was determined for three blots using NIH image, averaged and normalized to the intensity in wild-type plants at 14 dap. (C) GUS activity in the 3rd and 4th leaf of primary transgenic plants transformed with 35S::GUS-SPL3 (G-SPL3) or 35S::GUS-SPL3m (G-SPL3m). More than 30 plants of each genotype were analyzed. The difference between the expression of these constructs in wild type and zip-1 is not statistically significant. (D) Northern blot of SPL4, SPL9 and SPL15 mRNA in flowers of wild-type, sgs3-11 and rdr6-11 plants. (E) RT-PCR analysis of the SPL3 antisense transcript (SPL3 AS).