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, 133 (18), 3539-47

Temporal Regulation of Shoot Development in Arabidopsis Thaliana by miR156 and Its Target SPL3

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Temporal Regulation of Shoot Development in Arabidopsis Thaliana by miR156 and Its Target SPL3

Gang Wu et al. Development.

Abstract

SPL3, SPL4 and SPL5 (SPL3/4/5) are closely related members of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE family of transcription factors in Arabidopsis, and have a target site for the microRNA miR156 in their 3' UTR. The phenotype of Arabidopsis plants constitutively expressing miR156-sensitive and miR156-insensitive forms of SPL3/4/5 revealed that all three genes promote vegetative phase change and flowering, and are strongly repressed by miR156. Constitutive expression of miR156a prolonged the expression of juvenile vegetative traits and delayed flowering. This phenotype was largely corrected by constitutive expression of a miR156-insensitive form of SPL3. The juvenile-to-adult transition is accompanied by a decrease in the level of miR156 and an increase in the abundance of SPL3 mRNA. The complementary effect of hasty on the miR156 and SPL3 transcripts, as well as the miR156-dependent temporal expression pattern of a 35S::GUS-SPL3 transgene, suggest that the decrease in miR156 is responsible for the increase in SPL3 expression during this transition. SPL3 mRNA is elevated by mutations in ZIPPY/AGO7, RNA DEPENDENT RNA POLYMERASE 6 (RDR6) and SUPPRESSOR OF GENE SILENCING 3 (SGS3), indicating that it is directly or indirectly regulated by RNAi. However, our results indicate that RNAi does not contribute to the temporal expression pattern of this gene. We conclude that vegetative phase change in Arabidopsis is regulated by an increase in the expression of SPL3 and probably also SPL4 and SPL5, and that this increase is a consequence of a decrease in the level of miR156.

Figures

Fig. 1
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.
Fig. 2
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, SPL3Δ) 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 Δ 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).
Fig. 3
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.
Fig. 4
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 (Δ) a 3′ UTR. Error bars=s.e.m. (C,D) Morphology of the transgenic lines illustrated in B.
Fig. 5
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.
Fig. 6
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).

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