Genetic framework for flowering-time regulation by ambient temperature-responsive miRNAs in Arabidopsis

Abstract

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 degrees 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.

Figure 1.

Outline of the identification of the ambient temperature-responsive miRNAs in Arabidopsis. The miRNAs consistently identified by both the miRNA microarray and northern hybridization analyses were grouped and named as ambient temperature-responsive miRNAs.

Figure 2.

miRNA northern hybridization analysis of unique Arabidopsis miRNA loci with two biological replicates. The numbers above the blots indicate the times of harvest (ZT). The numbers below each miRNA blot denote fold change relative to the miRNA level at 23°C. Ethidium bromide-stained rRNAs are shown to demonstrate an equal amount of loading. Note that a probe hybridizes to its paralogous loci as well as itself, because of their identical sequences. The miRNA loci showing up-regulation at 16°C (A) and 23°C (B) are shown.

Figure 3.

Expression of the six ambient temperature-responsive miRNAs and their target genes at 23 and 16°C. Total RNAs isolated from 10-day-old wild-type seedlings and the same RNA were used for miRNA northern hybridization and RT–PCR of the target genes. The RT–PCR results are presented under the respective panels of the miRNA expression. (A) Expression of miR156 and miR169, showing up-regulation at 16°C, and the transcript levels of their target genes. (B) Expression of miR163, miR172, miR398 and miR399, showing up-regulation at 23°C, and the transcript levels of their target genes. The numbers below the miRNA blots denote fold change relative to the miRNA level at 23°C. The target genes whose cleavage site was validated by 5′ Rapid amplification of complementary DNA ends (RACE-PCR) analysis (8,43,44,58–60) or target genes whose expression was down-regulated in transgenic plants overexpressing miRNA (44,61) are indicated by an asterisk. The numbers to the right of every panel indicate the number of PCR cycles. U6 RNA and UBQ10 were used for a control for northern hybridization and RT–PCR analyses, respectively.

Figure 4.

(A) Expression of six ambient temperature-responsive miRNAs in the thermosensory pathway mutants at 23 and 16°C. miRNA accumulation in the fve-3, fca-9 and svp-32 mutants grown for 10 days was measured by northern hybridization. Ethidium bromide-stained rRNAs were used as loading controls. The numbers below the blots denote the fold change relative to wild-type plants. (B) qRT–PCR analysis of the target genes of miR172 in 10-day-old svp-32 mutants grown under LD conditions at 23 and 16°C. Tubulin was used to normalize the expression of target genes. Error bars indicate standard deviation.

Figure 5.

Changes in the ambient temperature response by miR172 overexpression. Phenotype (A) and flowering time (C) of miR172a-overexpressing plants (miR172a OX) grown under LD conditions at 23 and 16°C. The error bars denote the standard deviation. (B) miRNA northern hybridization to show overproduction of miR172 in miR172a OX plants grown at 23 and 16°C. (D) qRT–PCR analysis of the target genes of miR172 in 8-day-old miR172a OX plants grown under LD conditions at 23 and 16°C. (E) qRT–PCR analysis of flowering-time genes in 8-day-old miR172a OX plants grown under LD conditions at 23 and 16°C.