The sequential action of miR156 and miR172 regulates developmental timing in Arabidopsis

Abstract

The transition from the juvenile to the adult phase of shoot development in plants is accompanied by changes in vegetative morphology and an increase in reproductive potential. Here, we describe the regulatory mechanism of this transition. We show that miR156 is necessary and sufficient for the expression of the juvenile phase, and regulates the timing of the juvenile-to-adult transition by coordinating the expression of several pathways that control different aspects of this process. miR156 acts by repressing the expression of functionally distinct SPL transcription factors. miR172 acts downstream of miR156 to promote adult epidermal identity. miR156 regulates the expression of miR172 via SPL9 which, redundantly with SPL10, directly promotes the transcription of miR172b. Thus, like the larval-to-adult transition in Caenorhabditis elegans, the juvenile-to-adult transition in Arabidopsis is mediated by sequentially operating miRNAs. miR156 and miR172 are positively regulated by the transcription factors they target, suggesting that negative feedback loops contribute to the stability of the juvenile and adult phases.

Figure 1. miR156 is necessary and sufficient for the juvenile vegetative phase

(A) 25-day-old wild-type, 35S∷miR156a and 35S∷MIMI156 plants grown in short days. (B) The shape and the abaxial trichome phenotypes of fully expanded rosette leaves of wild type, 35S∷miR156a and 35S∷MIMI156 plants. 35S∷miR156a prolongs the duration of the juvenile phase and 35S∷MIM156 eliminates this phase. Asterisks indicate significant difference from wild-type (P < 0.01, n = 18, ± SD).

Figure 2. SPL3, SPL9 and SPL10 have diverse roles in vegetative development

(A–C) In situ expression pattern of SPL3. (A) 22 day-old vegetative shoot apex hybridized with a sense strand control. (B) 15-day-old vegetative shoot apex hybridized with antisense probe. (C) 22-day-old vegetative shoot apex hybridized with antisense probe. The abundance of SPL3 mRNA increases with time. (D–F) In situ expression pattern of SPL9; all samples hybridized with an antisense probe. (D) 22-day-old vegetative shoot apex of an RNA-null allele of SPL9. (E) 22-day-old wild-type vegetative shoot apex. (F) 22-day-old vegetative shoot apex from a plant expressing a miR156-insensitive SPL9 genomic sequence under the control of the SPL9 promoter. SPL9 is expressed in young leaf primordia. (G) Four-week-old rosettes of wild-type, mutant and transgenic lines of Arabidopsis grown in short days. rSPL3 = 35S∷rSPL3, rSPL9 = pSPL9∷rSPL9, rSPL10 = rSPL10∷rSPL10. (H) The shape and abaxial trichome phenotypes of fully expanded leaves of wild-type, spl9-4, and transgenic lines expressing miR156-resistant forms of SPL3, SPL9 and SPL10. These genes promote different adult traits. Asterisks indicate significant difference from wild-type (P <0.01, n= 18, ± SD).

Figure 3. miR172 acts downstream of miR156

(A) Blot of small RNA from the shoot apex of wild-type plants of different ages hybridized sequentially with probes to miR156 and miR172. The levels of these miRNAs change in a complementary fashion. U6 served as a loading control. (B) Blots of small RNA from 35S∷miR156a 35S∷miR172b (14-day-old) and 35S∷MIM156 (20-day-old) plants hybridized sequentially with probes to miR156 and miR172. miR156 represses miR172. U6 was used as loading control. (C) Leaf shape and abaxial trichome phenotypes of fully expanded rosette leaves of wild-type, 35S∷miR156a 35S∷miR172b and 35S∷miR156a 35S∷miR172b double transgenic plants. 35S∷miR172b partially rescues the 35S∷miR156a over-expression phenotype. Numbers indicate fold change relative to wild-type. Asterisks indicate significant difference from wild-type (P <0.01, n=18 plants, ± SD).

Figure 4. SPL3, SPL4 and SPL5 act independently of miR172 and TOE1, TOE2

(A) RNA blots of small RNA from 20-day-old wild-type, 35S∷rSPL3, 35S∷rSPL4 and 35S∷rSPL5 rosettes hybridized with a probe to miR172 reveals that these transgenes have no effect on the expression of miR172. U6 served as a loading control. Numbers indicate fold change relative to wild-type. (B) qRT-PCR analysis of TOE1 and TOE2 mRNA from 14-day-old wild-type, 35S∷rSPL3 35S∷rSPL4 and 35S∷rSPL5 rosettes indicates that these transgenes have no effect on the expression of TOE1 and TOE2. (C) qRT-PCR analysis of SPL3 mRNA in wild-type, toe1 toe2, and 35S∷TOE1 rosettes reveals that the expression of SPL3 is increased by toe1 toe2, but unaffected by 35S∷TOE1. (D) qRT-PCR analysis of SPL4 and SPL5 mRNA in 2-week-old wild-type and toe1 toe2 rosettes reveals no consistent change in the expression of these genes; the results of two experiments are shown. qRT-PCR data represent the average of three technical replicates; samples were normalized to wild-type at each time point; ± SD.

Figure 5. Regulation of miR172b by SPL9, SPL10, TOE1, and TOE2

(A) Northern blot of small RNA from 20-day-old wild-type, pSPL9∷rSPL9 and pSPL10∷rSPL10 rosettes. U6 was used as a loading control. Numbers indicate the fold change relative to wild-type. pSPL9∷rSPL9 and pSPL10∷rSPL10 increase the expression of miR172. (B) qRT-PCR analysis of the miR17b precursor in 12, 16, 19 and 24-day-old wild type, pSPL9∷rSPL9 pSPL10∷rSPL10 rosettes. The fold change relative to the 12-day-old wild type sample is shown; SD bars are obscured by symbols. These transgenes increase the expression of miR172b and eliminate its temporal expression pattern. (C) qRT-PCR analysis of the miR17b precursor in 20-day-old 35S∷GR-rSPL9 seedlings treated with DEX in the absence or presence of CHX. GR-SPL9 promotes the expression of miR172b in the absence of protein synthesis. (D) The location of three putative SPL9 binding sites in the miR172 locus that were tested by ChIP analysis. Open box indicates the miR172b transcript. (E) qPCR analysis of putative SPL9 binding sites in the chromatin of 14-day-old pSPL9∷SPL9r–cMyc and pSPL9∷3XFLAG-SPL9r rosettes immunoprecipitated with an antibody to FLAG. The immunoprecipitated values were first normalized to the input values then divided by the pSPL9∷SPL9r–cMyc value to get a fold enrichment. The numbers represent the fold difference relative to pSPL9∷SPL9r–cMyc sample. Values are the average of two biological replicates. eIF4A was used as a negative control. (F) qRT-PCR analysis of the miR172b precursor in wild-type and spl9-4 spl15-1 rosettes at different stages of vegetative development. miR172b is slightly reduced at all of these stages. (G) Blot of small RNA from the rosette of 14-day-old wild-type and toe1 toe2 plants hybridized with a probe to miR172. U6 served as a loading control. Numbers indicate the fold change relative to the wild-type sample. (H) qRT-PCR analysis of the miR172b precursor in wild-type, toe1 toe2 and 35S∷TOE1 rosettes. (I) qRT-PCR analysis of the miR156a precursor in 12- and 16-day-old pSPL9∷rSPL9 and pSPL10∷rSPL10 plants. qRT-PCR data represent the average of three technical replicates; samples were normalized to wild-type at each time point; ± SD.

Figure 6. A model for the regulation of vegetative phase change by miR156 and miR172

Temporal changes in the level of miR156 and SPL proteins are illustrated by the shaded bars; time increases from left to right. We propose that miR156 coordinates the expression of several pathways by repressing the expression of SPL genes that act in these pathways. Each of these pathways controls different phase-specific traits, but have components in common (e.g. SPL9, SPL10) and may also share downstream targets. The relationship between TOE1 and TOE2 and SPL3, SPL4, SPL5 is unclear.