SERRATE: a new player on the plant microRNA scene

Abstract

MicroRNAs (miRNAs) function as sequence-specific guides that control gene expression by post-transcriptional gene silencing. Many miRNAs influence plant development by regulating the accumulation of transcripts that encode transcription factors. Mutants defective in miRNA accumulation, such as dcl1, hen1, hyl1 and ago1, have pleiotropic developmental phenotypes. The serrate-1 (se-1) mutant of Arabidopsis also shows a highly pleiotropic phenotype, which overlaps with the phenotypes of mutants defective in miRNA accumulation. Although it has been proposed that SERRATE (SE) functions specifically in miRNA-mediated repression of the leaf polarity genes PHABULOSA and PHAVOLUTA, microarray analysis shows upregulation of many genes known to be the targets of miRNAs in se-1. We show that SE is a general regulator of miRNA levels affecting the processing of primary miRNA to miRNA.

Figure 1

serrate-1 decreases the accumulation of mature microRNAs and increases the accumulation of primary microRNA transcripts. (A) Northern blotting of total RNA from wild-type and serrate-1 (se-1) seedlings. For all microRNAs (miRNAs) tested (miRJAW, miR171, miR168, miR156, miR163, miR167, miR165, miR164), the se-1 mutant showed a reduced level of miRNA accumulation compared with wild type. (B) Accumulation of short interfering RNA (siRNA) in se-1 compared with wild type. The se-1 mutation had little effect on the accumulation of these siRNAs. The corresponding U6 loading control is shown below each blot. (C) Reverse transcription–PCR assay comparing levels of primary miRNAs (pri-miRNAs) in wild type and se-1. PCR products were blotted and hybridized with random-primed probes. The UBIQUITIN (UBQ) loading control is shown below the blots.

Figure 2

serrate-1 decreases the accumulation of microRNA cleavage products. (A) Agarose gels showing the nested PCR products of PHB, NAC1 and ARF8 microRNA (miRNA) cleavage products. (B) Southern blot analysis of the PHB, NAC1 and ARF8 miRNA cleavage products hybridized with a probe corresponding to the 5′ rapid amplification of cloned ends nested PCR products. The UBIQUITIN (UBQ) loading control is shown below. ARF, AUXIN RESPONSE FACTOR; NAC, for NAM, ATAF1-2, CUC2; PHB, PHABULOSA; se-1, serrate-1; WT, wild type.

Figure 3

The serrate-4 mutant allele confers embryonic lethality. The development of wild-type embryos (A–C) and serrate-4 (se-4) mutant embryos (D–F) is shown. Each column depicts embryos from seeds of the same age. Scale bars, 50 μm. (A) Wild-type, heart-stage embryo with cotyledon primordia. (B) Wild-type, torpedo-stage embryos showing elongating cotyledons. (C) Wild-type, curled cotyledon-stage embryo. (D) se-4, heart-stage embryo. (E) se-4, torpedo-stage embryo. (F) se-4, curled cotyledon-stage embryo.