Sugar promotes vegetative phase change in Arabidopsis thaliana by repressing the expression of MIR156A and MIR156C
- University of Pennsylvania, United States
Figures
Figure 1
The prolonged juvenile phase of ch1 is suppressed by 35S::MIM156.
(A) Wild-type Col produces about six juvenile leaves (5.7 ± 0.9, N = 24) in SD conditions. (B) ch1-4 produces significantly more juvenile leaves (10.9 ± 0.8, N = 24, p<0.01) than Col. (C) 35S::MIM156. (D) ch1-4 35S::MIM156. This genotype has the morphology of 35S::MIM156 and the yellow-green phenotype of ch1-4; double mutants produced abaxial trichomes on leaf 1 and had elongated serrated leaves, demonstrating that the delayed phase change phenotype of ch1-4 is dependent on miR156. Numbers indicate the position from the base of the plant. Scale bar = 5 mm.
Figure 2
Expression of miR156 and SPL transcripts in ch1-4.
(A) Northern blot of mature miR156 in ch1-4 and Col reveals that miR156 is elevated in ch1-4 and declines at a slower rate in this mutant. U6 was used as a loading control. Hybridization intensities are compared to the value in WT 12 DAP. (B) qRT-PCR of the transcripts of SPL3, SPL9, and SPL13 in 16-day-old wild-type and ch1-4 demonstrates that these transcripts are present at a significantly lower level in ch1-4. (C) MUG assay of the GUS activity of miR156-sensitive and miR156-resistant SPL3 and SPL9 reporters in Col and ch1-4 demonstrates that the reduced expression of GUS-SPL3 and SPL9-GUS in ch1 is dependent on miR156. (D) The expression pattern of miR156-sensitive and miR156-resistant pSPL3::GUS-SPL3 in 14-day-old rosettes of Col and 18-day-old rosettes of ch1-4; older rosettes of ch1-4 were chosen to compensate for the slower growth rate of this mutant. pSPL3::GUS-SPL3 is expressed at a lower level in ch1-4. pSPL3::rSPL3-GUS is expressed at the same level and in the same pattern in Col and ch1-4. Scale bar = 2 mm. Asterisks in (B) and (C) indicate significant difference (Student's t-test), p<0.01, n = 3. Error bars indicate SEM.
Figure 3
Sugar represses miR156 expression.
(A) Northern blot of miR156 in 12-day-old plants treated with 10-mM glucose (Glc), fructose (Fru), mannitol (Man), sorbitol (Sor), and O-methyl-glucose (OMG). U6 was used as a loading control. Only Glc and Fru reduce miR156 expression. (B) Northern blot of miR156 in 12-day-old plants treated for 4 hr with 10 mM of the indicated substances. The effect of glucose on miR156 expression is blocked by cycloheximide, a protein synthesis inhibitor. Man: mannitol; Glc: glucose; CHX: cycloheximide. (C) Northern blot of miR156 in 12-day-old ch1-4 plants treated with different amounts of glucose. 0.5 mM produced a 50% reduction in miR156, and higher amounts of glucose did not produce a further reduction. (D) 5-mm primordia of leaf 6 from pSPL9::SPL9-GUS and pSPL9::rSPL9-GUS plants, cultured for 8 hr in media containing 10 mM of different sugars. Scale bar = 1 mm. (E) Exogenous sucrose significantly accelerates abaxial trichome production in wild-type plants. Asterisk indicates significant difference (p<0.01; n = 12; error bars indicate SEM).
Figure 4
MIR156A and MIR156C are important for vegetative phase change.
(A) qRT-PCR of primary miRNAs in 1-mm leaf primordia from different positions in se-1 shoots, counting from the base of the rosette. Only MIR156A and C are temporally expressed. (B) The genomic structure of MIR156A and C and the location of T-DNA insertions in these genes. Boxes indicate exons, with the position of the miR156 hairpin indicated in gray. Arrows indicate the transcription start sites for the major MIR156A transcripts. (C) Northern blot of miR156 in single and double mutants of MIR156A and MIR156C. These mutations only affect the accumulation of the 20-nt form of miR156, demonstrating that the 21-nt form is the product of another gene or genes. (D) Rosette leaves of wild-type and mir156a-1 mir156c-1 plants grown in LD. (E) The mir156a-1 mir156c-1 double mutant has significantly fewer juvenile leaves and transition/adult leaves (error bars indicate SEM). (F) The mir156a-1 mir156c-1 double mutant has a reduced rate of leaf initiation.
Figure 5 with 1 supplement
Sugar specifically regulates the expression of MIR156 genes that are important for vegetative phase change.
(A) qRT-PCR of primary MIRNAs in 12-day-old se-1 plants grown in the absence or presence of 10 mM glucose. MIR156A, C, F, and H are repressed by glucose. Asterisk indicates significantly different from the no sugar treatment, p<0.01. (B) The structure of MIR156A-GUS and MIR156C-GUS reporter constructs, and their response to 50 mM glucose or sucrose. Blue indicates the location of GUS, which was inserted in place of the miR156 hairpin. The full-length constructs are approximately 7 kb in length. Solid line: intergenic sequence; dashed line: intron; bar = 2 mm. The staining response was representative of two independent lines, homozygous for a single transgenic insertion. (C) qRT-PCR analysis of the abundance of the MIR156A-GUS, MIR156C-GUS, pri-MIR156A, and pri-MIR156C transcripts in the seedlings illustrated in (B). GUS-fusion transcripts were measured using primers specific for GUS+. Asterisk indicates significantly different from the no sugar treatment, p<0.01.
Figure 5—figure supplement 1
Sucrose represses the expression of the MIR156A-GUS and MIR156C-GUS reporter genes in transgenic seedlings.
Seedlings were stained for 1.5 hr in 2 mM X-gluc, 2 mM ferricyanide, and 2 mM ferro- cyanide. Scale bar = 2 mm.
Figure 6 with 2 supplements
The signaling role of HXK1 is required for glucose-mediated repression of miR156.
(A) Northern blot of miR156 in 8- and 11-day-old Ler and gin2-1 seedlings grown in soil. gin2-1 has less miR156 than wild-type seedlings 8 days after planting, but has normal levels of miR156 at later stages. Hybridization intensities are compared to the intensity in Ler, 8 DAP. (B) Northern blot of miR156 in Ler and gin2-1 grown in the absence and presence of glucose. gin2-1 has lower levels of miR156 than Ler in the absence of glucose and is not further repressed by exogenous glucose. (C) Northern blot of miR156 in gin2-1 plants transformed with a wild-type HXK1 construct and a construct (S177A) that lacks enzymatic activity. miR156 accumulation was repressed by glucose in these transgenics by approximately the same amount as in Ler. (D) The number of leaves without abaxial trichomes in Ler, gin2-1, HXK1/gin2-1, and S177A/gin2-1 grown under SD, 16°C, and a light intensity of 60 µmol/m2/s. gin2-1 has significantly fewer juvenile leaves than Ler and the transgenic lines (a, n = 48, p<0.01, error bars indicate SEM). The number of juvenile leaves in gin2-1 plants transformed with wild-type (HXK1) and enzymatically inactive (S177A) HXK1 was not significantly different from wild-type Ler (b, n = 48, p>0.1, error bars indicate SEM).
Figure 6—figure supplement 1
HXK1 promotes the accumulation of miR156 in the absence of exogenous sugar.
In a sugar-free medium, gin2-1 has less miR156 than Ler and less miR156 than gin2-1 plants expressing a wild-type (HXK1) or catalytically inactive (S177A) form of HXK1. 10 mM glucose decreases the level of miR156 in Ler and in HXK1/gin2-1 and S177A/gin2-1, but has no effect on miR156 in gin2-1.
Figure 6—figure supplement 2
The timing of abaxial trichome production in wild-type gin2-1 and gin2-1 plants transformed with a wild-type (HXK1) or catalytically inactive form (S177A) of HXK1.
Plants grown in soil in short days (10 hr light, 220 μmol/m2/s) at 22°C. a: significantly different from other genotypes (p<0.01); b: not significantly different from Ler (p>0.05); Error barsindicate SEM.
Figure 7
Glucose rescues the defoliation-induced increase in miR156.
(A) 3-week-old N. benthamiana plant with agarose gel on petiole stubs. Scale bar = 1 cm. (B) Northern analysis of the level of miR156 in the shoot apex of wounded nondefoliated plants (used as a control), and defoliated plants treated with leaf extract (Ext), or 300 mM of mannitol (Man) or glucose (Glc). miR156 was assayed 3 days after treatment.
Additional files
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Supplementary file 1
The genomic organization of MIR156A (At2g25095). The two most abundant transcripts are indicated. Exons are indicated in yellow. The miR156 hairpin is underlined, and the mature miRNA is indicated in blue.
- https://doi.org/10.7554/eLife.00260.013
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Supplementary file 2
The genomic organization of MIR156C (At4g31877). Exons are indicated in yellow. The miR156 hairpin is underlined, and the mature miRNA is indicated in blue. A variant 5′ end of the transcript is indicated in green.
- https://doi.org/10.7554/eLife.00260.014
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Supplementary file 3
PCR primers and oligonucleotide probes.
- https://doi.org/10.7554/eLife.00260.015
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Sugar promotes vegetative phase change in Arabidopsis thaliana by repressing the expression of MIR156A and MIR156C
eLife 2:e00260.
https://doi.org/10.7554/eLife.00260
