Abstract
Plant microRNAs (miRNAs) are small, regulatory non-coding RNAs involved in a wide range of biological processes, from organ development to response to stimuli. In recent years, an increasing number of studies on model plant species have highlighted the evolutionary conservation of a high number of miRNA families and the existence of taxon-specific ones. However, few studies have examined miRNAs in non-model species such as orchids, which are characterized by highly diversified floral structures and pollination strategies. Therefore, we analysed a small RNA library of inflorescence tissue of the Mediterranean orchid Orchis italica to increase the knowledge on miRNAs in a non-model plant species. The high-throughput sequencing and analysis of a small RNA library of inflorescence of O. italica revealed 23 conserved and 161 putative novel miRNA families. Among the putative miRNA targets, experimental validation demonstrated that a DEF-like MADS-box transcript is cleaved by the homolog of miR5179 of O. italica. The presence of conserved miRNA families in the inflorescence of O. italica indicates that the basic developmental flower regulatory mechanisms mediated by miRNAs are maintained through evolution. Because, according to the “orchid code” theory, DEF-like genes exert a key function in the diversification of tepals and lip, the cleavage-mediated inhibitory activity of miR5179 on a OitaDEF-like transcript suggests that, in orchids, miRNAs play an important role in the diversification of the perianth organs.
Highlights
MicroRNAs are small (18–25 nt) non-coding RNAs that regulate gene expression at post-transcriptional level
Within the specified length range, the number of reads was highest between 21–24 nucleotides, with a peak at 24 nt (Figure 1). This is in agreement with the distribution pattern of small RNAs in other plant species such as Arabidopsis thaliana [37], Caragana intermedia [8], Solanum tuberosum [38], Avicennia marina [7], Oryza sativa [39] and the orchid P. aphrodite [10]
Using the newly assembled transcriptome of P. aphrodite as reference, we looked for novel miRNAs using miRDeep-P, and identified 478 distinct sequences of inflorescence that matched the criteria of plant miRNA structures
Summary
MicroRNAs (miRNAs) are small (18–25 nt) non-coding RNAs that regulate gene expression at post-transcriptional level. They originate from single-stranded precursors (pre-miRNAs) able to self-pair and form hairpin structures. The Dicer1-like RNAse III enzyme excises the miRNA/miRNA* duplex from the pre-miRNA. The guide strand of the duplex is recruited by an RNA-induced silencing complex (RISC), whose core component is the protein ARGONAUTE1 (AGO1). This complex permits the interaction between the miRNA and its target mRNA, thereby regulating its expression through mRNA cleavage, translational repression or epigenetic modifications [1,2]. Plant miRNAs play crucial roles in different processes, from leaf and root morphogenesis to floral induction, organ formation, reproduction and stress response [2,3,4,5]
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