Open Access Research article

Evolution of MIR168 paralogs in Brassicaceae

Silvia Gazzani1, Mingai Li1, Silvia Maistri2, Eliana Scarponi3, Michele Graziola1, Enrico Barbaro1, Jörg Wunder1, Antonella Furini2, Heinz Saedler4 and Claudio Varotto1*

Author Affiliations

1 Environment and Natural Resources Area, Fondazione Edmund Mach, via Mach 1, 38010 San Michele all'Adige (TN), Italy

2 Department of Science and Technology, University of Verona, strada le Grazie 15, 37134 Verona, Italy

3 Immuno-hematology and Transfusion Service, Santa Chiara Hospital, Largo Medaglie D'Oro 9, 38100 Trento, Italy

4 Department of Molecular Plant Genetics, Max Planck Institute for Plant Breeding Research, Carl-von-Linné-Weg 10, 50829 Cologne, Germany

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BMC Evolutionary Biology 2009, 9:62  doi:10.1186/1471-2148-9-62

Published: 23 March 2009



In plants, expression of ARGONAUTE1 (AGO1), the catalytic subunit of the RNA-Induced Silencing Complex responsible for post-transcriptional gene silencing, is controlled through a feedback loop involving the miR168 microRNA. This complex auto-regulatory loop, composed of miR168-guided AGO1-catalyzed cleavage of AGO1 mRNA and AGO1-mediated stabilization of miR168, was shown to ensure the maintenance of AGO1 homeostasis that is pivotal for the correct functioning of the miRNA pathway.


We applied different approaches to studying the genomic organization and the structural and functional evolution of MIR168 homologs in Brassicaeae. A whole genome comparison of Arabidopsis and poplar, phylogenetic footprinting and phylogenetic reconstruction were used to date the duplication events originating MIR168 homologs in these genomes. While orthology was lacking between Arabidopsis and poplar MIR168 genes, we successfully isolated orthologs of both loci present in Arabidopsis (MIR168a and MIR168b) from all the Brassicaceae species analyzed, including the basal species Aethionema grandiflora, thus indicating that (1) independent duplication events took place in Arabidopsis and poplar lineages and (2) the origin of MIR168 paralogs predates both the Brassicaceae radiation and the Arabidopsis alpha polyploidization. Different phylogenetic footprints, corresponding to known functionally relevant regions (transcription starting site and double-stranded structures responsible for microRNA biogenesis and function) or for which functions could be proposed, were found to be highly conserved among MIR168 homologs. Comparative predictions of the identified microRNAs also indicate extreme conservation of secondary structure and thermodynamic stability.


We used a comparative phylogenetic footprinting approach to identify the structural and functional constraints that shaped MIR168 evolution in Brassicaceae. Although their duplication happened at least 40 million years ago, we found evidence that both MIR168 paralogs have been maintained throughout the evolution of Brassicaceae, most likely functionally as indicated by the extremely high conservation of functionally relevant regions, predicted secondary structure and thermodynamic profile. Interestingly, the expression patterns observed in Arabidopsis indicate that MIR168b underwent partial subfunctionalization as determined by the experimental characterization of its expression pattern provided in this study. We found further evolutionary evidence that pre-miR168 lower stem (the RNA-duplex structure adjacent to the miR-miR* stem) is significantly longer than animal lower stems and probably plays a relevant role in multi-step miR168 biogenesis.