Abstract
Background: Winter wheat requires prolonged exposure to low temperature to initiate flowering (vernalization). Shoot apical meristem of the crown is the site of cold perception, which produces leaf primordia during vegetative growth before developing into floral primordia at the initiation of the reproductive phase. Although many essential genes for winter wheat cold acclimation and floral initiation have been revealed, the importance of microRNA (miRNA) meditated post-transcriptional regulation in crowns is not well understood. To understand the potential roles of miRNAs in crown tissues, we performed a temporal expression study of miRNAs in crown tissues at the three-leaf stage, winter dormancy stage, spring green-up stage, and jointing stage of winter wheat grown under natural growth conditions. Results: In total, 348 miRNAs belonging to 298 miRNA families, were identified in wheat crown tissues. Among them, 92 differentially expressed miRNAs (DEMs) were found to be significantly regulated from the three-leaf stage to the jointing stage. Most of these DEMs were highly expressed at the three-leaf stage and winter dormancy stage, and then declined in later stages. Six DEMs, including miR156a-5p were markedly induced during the winter dormancy stage. Eleven DEMs, including miR159a.1, miR390a-5p, miR393-5p, miR160a-5p, and miR1436, were highly expressed at the green-up stage. Twelve DEMs, such as miR172a-5p, miR394a, miR319b-3p, and miR9676-5p were highly induced at the jointing stage. Moreover, 14 novel target genes of nine wheat or Pooideae-specific miRNAs were verified using RLM-5' RACE assay. Notably, six mTERFs and two Rf1 genes, which are associated with mitochondrial gene expression, were confirmed as targets of three wheat-specific miRNAs. Conclusions: The present study not only confirmed the known miRNAs associated with phase transition and floral development, but also identified a number of wheat or Pooideae-specific miRNAs critical for winter wheat cold acclimation and floral development. Most importantly, this study provided experimental evidence that miRNA could regulate mitochondrial gene expression by targeting mTERF and Rf1 genes. Our study provides valuable information for further exploration of the mechanism of miRNA mediated post-transcriptional regulation during winter wheat vernalization and inflorescent initiation.