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High-throughput sequencing of RNA silencing-associated small RNAs in olive (Olea europaea L.).

ABSTRACT: Small RNAs (sRNAs) of 20 to 25 nucleotides (nt) in length maintain genome integrity and control gene expression in a multitude of developmental and physiological processes. Despite RNA silencing has been primarily studied in model plants, the advent of high-throughput sequencing technologies has enabled profiling of the sRNA component of more than 40 plant species. Here, we used deep sequencing and molecular methods to report the first inventory of sRNAs in olive (Olea europaea L.). sRNA libraries prepared from juvenile and adult shoots revealed that the 24-nt class dominates the sRNA transcriptome and atypically accumulates to levels never seen in other plant species, suggesting an active role of heterochromatin silencing in the maintenance and integrity of its large genome. A total of 18 known miRNA families were identified in the libraries. Also, 5 other sRNAs derived from potential hairpin-like precursors remain as plausible miRNA candidates. RNA blots confirmed miRNA expression and suggested tissue- and/or developmental-specific expression patterns. Target mRNAs of conserved miRNAs were computationally predicted among the olive cDNA collection and experimentally validated through endonucleolytic cleavage assays. Finally, we use expression data to uncover genetic components of the miR156, miR172 and miR390/TAS3-derived trans-acting small interfering RNA (tasiRNA) regulatory nodes, suggesting that these interactive networks controlling developmental transitions are fully operational in olive.

SUBMITTER: Donaire L

PROVIDER: S-EPMC3225373 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The ripening process of olive fruits is associated with chemical and/or enzymatic specific transformations making them particularly attractive to animals and humans. Usually, there is a progressive chromatic change characterized by a final red-brown color of both epidermis and mesocarp. This event has an exception in the ‘Leucocarpa’ cultivar, in which we observed the destabilization in the equilibrium between the metabolism on chlorophyll and that of the other pigments, particularly the anthocyanins, whose switch-off during maturation promotes the white coloration of the fruits. Recently, transcription profiling of ripening olives from ‘Leucocarpa’ and ‘Cassanese’ cultivars, through an Illumina RNA-seq approach, has provided some information about genes involved in fruit maturation as flavonid and anthocyanin transcripts. To study the possible gene differences involved in flavonoids and anthocyanins biosynthetic pathways during ripening possibly caused by small nuclear RNA (snRNA) in olive drupes, the snRNA libraries of fruit ripening from ‘Leucocarpa’ and ‘Cassanese’ were constructed with RNAs from drupes at 100 and 130 DAF (Days After Flowering) and sequenced by Illumina approach. Comparing sequence information, 130 conserved microRNAs (miRNA) in the Viridiplantae were detected belonging 14 miRNA families. For the remaining read not-matched with known miRNAs in the Viridiplantae, we combined secondary structure and minimum free energy to discover novel olive miRNAs. Based on these analyses, 492 miRNAs were considered as putative novel. To increase insights into the functions of known and putative novel miRNA in olive, putative target genes were computationally predicted by alignment with the olive drupe transcripts obtained from the same samples. A total of 32 and 186 transcripts were predicted as targets of 130 known and 492 putative novel miRNA, respectively. The identified target genes are involved in a broad range of biological processes. Interestingly, some genes involved in negative regulation of anthocyanin metabolic process, were identified as target of miR168 suggesting that this miRNA family is likely operative during color transition in olive.

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High-throughput sequencing of RNA silencing-associated small RNAs in olive (Olea europaea L.).

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