Flowering is the primary trait affected by ambient temperature changes. Plant microRNAs (miRNAs) are small non-coding RNAs playing an important regulatory role in plant development. In this study, to elucidate the mechanism of flowering-time regulation by small RNAs, we identified six ambient temperature-responsive miRNAs (miR156, miR163, miR169, miR172, miR398 and miR399) in Arabidopsis via miRNA microarray and northern hybridization analyses. We also determined the expression profile of 120 unique miRNA loci in response to ambient temperature changes by miRNA northern hybridization analysis. The expression of the ambient temperature-responsive miRNAs and their target genes was largely anticorrelated at two different temperatures (16 and 23°C). Interestingly, a lesion in short vegetative phase (SVP), a key regulator within the thermosensory pathway, caused alteration in the expression of miR172 and a subset of its target genes, providing a link between a thermosensory pathway gene and miR172. The miR172-overexpressing plants showed a temperature-independent early flowering phenotype, suggesting that modulation of miR172 expression leads to temperature insensitivity. Taken together, our results suggest a genetic framework for flowering-time regulation by ambient temperature-responsive miRNAs under non-stress temperature conditions.

The flowering time of plants is affected by modest changes in ambient temperature. However, little is known about the regulation of ambient temperature-responsive flowering by small RNAs. In this study, we show that the microRNA156 (miR156)-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 (SPL3) module directly regulates FLOWERING LOCUS T (FT) expression in the leaf to control ambient temperature-responsive flowering. Overexpression of miR156 led to more delayed flowering at a lower ambient temperature (16°C), which was associated with down-regulation of FT and FRUITFULL expression. Among miR156 target genes, SPL3 mRNA levels were mainly reduced, probably because miR156-mediated cleavage of SPL3 mRNA was higher at 16°C. Overexpression of miR156-resistant SPL3 [SPL3(2)] caused early flowering, regardless of the ambient temperature, which was associated with up-regulation of FT and FRUITFULL expression. Reduction of miR156 activity by target mimicry led to a phenotype similar to that of SUC2::rSPL3 plants. FT up-regulation was observed after dexamethasone treatment in GVG-rSPL3 plants. Misexpression and artificial microRNA-mediated suppression of FT in the leaf dramatically altered the ambient temperature-responsive flowering of plants overexpressing miR156 and SPL3(2). Chromatin immunoprecipitation assay showed that the SPL3 protein directly binds to GTAC motifs within the FT promoter. Lesions in TERMINAL FLOWER1, SHORT VEGETATIVE PHASE, and EARLY FLOWERING3 did not alter the expression of miR156 and SPL3. Taken together, our data suggest that the interaction between the miR156-SPL3 module and FT is part of the regulatory mechanism controlling flowering time in response to ambient temperature.

Background: Histone acetyltransferases (HATs) play critical roles in the regulation of chromatin structure and gene expression. Arabidopsis genome contains 12 HAT genes, but the biological functions of many of them are still unknown. In this work, we studied the evolutionary relationship and cellular functions of the two Arabidopsis HAT genes homologous to the MYST family members.

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A moderate change in ambient temperature significantly affects plant physiology including flowering time. MiR399 and its target gene PHOSPHATE 2 (PHO2) are known to play a role in the maintenance of phosphate homeostasis. However, the regulation of flowering time by the miR399-PHO2 module has not been investigated. As we have previously identified miR399 as an ambient temperature-responsive miRNA, we further investigated whether a change in expression of the miR399-PHO2 module affects flowering time in response to ambient temperature changes. Here, we showed that miR399b-overexpressing plants and a loss-of-function allele of PHO2 (pho2) exhibited an early flowering phenotype only at normal temperature (23°C). Interestingly, their flowering time at lower temperature (16°C) was similar to that of wild-type plants, suggesting that alteration in flowering time by miR399 and its target PHO2 was seen only at normal temperature (23°C). Flowering time ratio (16°C/23°C) revealed that miR399b-overexpressing plants and pho2 mutants showed increased sensitivity to ambient temperature changes. Expression analysis indicated that expression of TWIN SISTER OF FT (TSF) was increased in miR399b-overexpressing plants and pho2 mutants at 23°C, suggesting that their early flowering phenotype is associated with TSF upregulation. Taken together, our results suggest that miR399, an ambient temperature-responsive miRNA, plays a role in ambient temperature-responsive flowering in Arabidopsis.

MicroRNAs (miRNA) that guide sequence-specific posttranscriptional gene silencing play an important role in gene expression required for both developmental processes and responses to environmental conditions in plants. However, little is known about the transcriptional and posttranscriptional regulation of miRNA expression. Histone acetylation plays an important role in chromatin remodeling and is required for gene activation. By analyzing the accumulation of subset of miRNAs and the corresponding primary miRNAs in mutants of Arabidopsis, we show that histone acetyltransferase GCN5 (General control non-repressed protein5) has a general repressive effect on miRNA production, while it is required for the expression of a subset of (e.g. stress-inducible) MIRNA genes. The general negative function of GCN5 in miRNA production is likely achieved through an indirect repression of the miRNA machinery genes such as DICER LIKE1 (DCL1), SERRATE (SE), HYPONASTIC LEAVES1 (HYL1) and ARGONAUTE1 (AGO1). Chromatin immunoprecipitation assays revealed that GCN5 targets to a subset of MIRNA genes and is required for acetylation of histone H3 lysine 14 at these loci. Moreover, inhibition of histone deacetylation by trichostatin A treatment or in histone deacetylase gene mutants impaired the accumulation of certain miRNAs. These data together suggest that Arabidopsis GCN5 interferes with the miRNA pathway at both the transcriptional and posttranscriptional levels and histone acetylation/deacetylation is an epigenetic mechanism involved in the regulation of miRNA production.

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.

a b s t r a c tPlant microRNAs (miRNAs) are non-coding RNAs that negatively regulate expression of their target genes. Although much is known about miRNA biogenesis and repression of target genes by miRNAs, the molecular mechanisms underlying the transcriptional regulation of miRNA itself are poorly understood. Here, we report that SHORT VEGETATIVE PHASE (SVP) protein is a direct transcriptional regulator of miR172. The levels of mature miR172 and pri-miR172a were anti-correlated with SVP activity. miR172a has multiple transcription start sites, among which the transcript starting with cytosine (À671 bp, relative to the mature miR172a) was a major species. EMSA and ChIP analysis demonstrated that SVP protein binds to the CArG motifs in the miR172a promoter. These results suggest that SVP protein directly regulates miR172 transcription in Arabidopsis. Crown