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Expression of the floral repressor miRNA156 is positively regulated by the AGAMOUS-like proteins AGL15 and AGL18.

Serivichyaswat P, Ryu HS, Kim W, Kim S, Chung KS, Kim JJ, Ahn JH - Mol. Cells (2015)

Bottom Line: Small RNA northern blot analysis revealed a significant decrease in the levels of mature miR156 in agl15 agl18 double mutants, but not in the single mutants, suggesting that AGL15 and AGL18 co-regulate miR156 expression.AGL18 did not show binding affinity to the CArG motifs, but pull-down and yeast two-hybrid assays showed that AGL18 forms a heterodimer with AGL15.Overexpression of miR156 did not affect the levels of AGL15 and AGL18 transcripts.

View Article: PubMed Central - PubMed

Affiliation: Creative Research Initiatives, Department of Life Sciences, Korea University, Seoul 136-701, Korea.

ABSTRACT
The regulation of flowering time has crucial implications for plant fitness. MicroRNA156 (miR156) represses the floral transition in Arabidopsis thaliana, but the mechanisms regulating its transcription remain unclear. Here, we show that two AGAMOUS-like proteins, AGL15 and AGL18, act as positive regulators of the expression of MIR156. Small RNA northern blot analysis revealed a significant decrease in the levels of mature miR156 in agl15 agl18 double mutants, but not in the single mutants, suggesting that AGL15 and AGL18 co-regulate miR156 expression. Histochemical analysis further indicated that the double mutants showed a reduction in MIR156 promoter strength. The double mutants also showed reduced abundance of pri-miR156a and pri-miR156c, two of the primary transcripts from MIR156 genes. Electrophoretic mobility shift assays demonstrated that AGL15 directly associated with the CArG motifs in the MIR156a/c promoters. AGL18 did not show binding affinity to the CArG motifs, but pull-down and yeast two-hybrid assays showed that AGL18 forms a heterodimer with AGL15. GFP reporter assays and bimolecular fluorescence complementation (BiFC) showed that AGL15 and AGL18 co-localize in the nucleus and confirmed their in vivo interaction. Overexpression of miR156 did not affect the levels of AGL15 and AGL18 transcripts. Taking these data together, we present a model for the transcriptional regulation of MIR156. In this model, AGL15 and AGL18 may form a complex along with other proteins, and bind to the CArG motifs of the promoters of MIR156 to activate the MIR156 expression.

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Relative levels of primary transcripts of miR156a, c, and d by qPCR. The levels of each primary transcript in wild-type Col-0 plants were set to one.
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f2-molcell-38-3-259: Relative levels of primary transcripts of miR156a, c, and d by qPCR. The levels of each primary transcript in wild-type Col-0 plants were set to one.

Mentions: The mature miR156 is produced from primary transcripts of miR156 (pri-miR156) from eight different loci (MIR156a-h); therefore, we investigated which locus is mainly affected in the agl15 agl18 double mutants. Absolute quantification of the levels of the primary transcripts revealed that pri-miR156a, pri-miR156c, and pri-miR156d are the main loci that produce miR156 in wild-type plants (data not shown). Thus we measured their transcript levels in wild-type and agl15 agl18 double mutant plants. The expression of pri-miR156a and pri-miR156c were reduced in the double mutants, compared to the wild-type plants (Fig. 2), whereas pri-miR156d levels showed no significant changes in either of the double mutant lines (Fig. 2). This result suggested that AGL15 and AGL18 mainly regulate the transcription of MIR156a and MIR156c. Therefore, we focused on miR156a and miR156c for the subsequent studies.


Expression of the floral repressor miRNA156 is positively regulated by the AGAMOUS-like proteins AGL15 and AGL18.

Serivichyaswat P, Ryu HS, Kim W, Kim S, Chung KS, Kim JJ, Ahn JH - Mol. Cells (2015)

Relative levels of primary transcripts of miR156a, c, and d by qPCR. The levels of each primary transcript in wild-type Col-0 plants were set to one.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4363726&req=5

f2-molcell-38-3-259: Relative levels of primary transcripts of miR156a, c, and d by qPCR. The levels of each primary transcript in wild-type Col-0 plants were set to one.
Mentions: The mature miR156 is produced from primary transcripts of miR156 (pri-miR156) from eight different loci (MIR156a-h); therefore, we investigated which locus is mainly affected in the agl15 agl18 double mutants. Absolute quantification of the levels of the primary transcripts revealed that pri-miR156a, pri-miR156c, and pri-miR156d are the main loci that produce miR156 in wild-type plants (data not shown). Thus we measured their transcript levels in wild-type and agl15 agl18 double mutant plants. The expression of pri-miR156a and pri-miR156c were reduced in the double mutants, compared to the wild-type plants (Fig. 2), whereas pri-miR156d levels showed no significant changes in either of the double mutant lines (Fig. 2). This result suggested that AGL15 and AGL18 mainly regulate the transcription of MIR156a and MIR156c. Therefore, we focused on miR156a and miR156c for the subsequent studies.

Bottom Line: Small RNA northern blot analysis revealed a significant decrease in the levels of mature miR156 in agl15 agl18 double mutants, but not in the single mutants, suggesting that AGL15 and AGL18 co-regulate miR156 expression.AGL18 did not show binding affinity to the CArG motifs, but pull-down and yeast two-hybrid assays showed that AGL18 forms a heterodimer with AGL15.Overexpression of miR156 did not affect the levels of AGL15 and AGL18 transcripts.

View Article: PubMed Central - PubMed

Affiliation: Creative Research Initiatives, Department of Life Sciences, Korea University, Seoul 136-701, Korea.

ABSTRACT
The regulation of flowering time has crucial implications for plant fitness. MicroRNA156 (miR156) represses the floral transition in Arabidopsis thaliana, but the mechanisms regulating its transcription remain unclear. Here, we show that two AGAMOUS-like proteins, AGL15 and AGL18, act as positive regulators of the expression of MIR156. Small RNA northern blot analysis revealed a significant decrease in the levels of mature miR156 in agl15 agl18 double mutants, but not in the single mutants, suggesting that AGL15 and AGL18 co-regulate miR156 expression. Histochemical analysis further indicated that the double mutants showed a reduction in MIR156 promoter strength. The double mutants also showed reduced abundance of pri-miR156a and pri-miR156c, two of the primary transcripts from MIR156 genes. Electrophoretic mobility shift assays demonstrated that AGL15 directly associated with the CArG motifs in the MIR156a/c promoters. AGL18 did not show binding affinity to the CArG motifs, but pull-down and yeast two-hybrid assays showed that AGL18 forms a heterodimer with AGL15. GFP reporter assays and bimolecular fluorescence complementation (BiFC) showed that AGL15 and AGL18 co-localize in the nucleus and confirmed their in vivo interaction. Overexpression of miR156 did not affect the levels of AGL15 and AGL18 transcripts. Taking these data together, we present a model for the transcriptional regulation of MIR156. In this model, AGL15 and AGL18 may form a complex along with other proteins, and bind to the CArG motifs of the promoters of MIR156 to activate the MIR156 expression.

Show MeSH