Sequencing of small RNAs of the fern Pleopeltis minima (Polypodiaceae) offers insight into the evolution of the microrna repertoire in land plants

Abstract

MicroRNAs (miRNAs) are short, single stranded RNA molecules that regulate the stability and translation of messenger RNAs in diverse eukaryotic groups. Several miRNA genes are of ancient origin and have been maintained in the genomes of animal and plant taxa for hundreds of millions of years, playing key roles in development and physiology. In the last decade, genome and small RNA (sRNA) sequencing of several plant species have helped unveil the evolutionary history of land plants. Among these, the fern group (monilophytes) occupies a key phylogenetic position, as it represents the closest extant cousin taxon of seed plants, i.e. gymno- and angiosperms. However, in spite of their evolutionary, economic and ecological importance, no fern genome has been sequenced yet and few genomic resources are available for this group. Here, we sequenced the small RNA fraction of an epiphytic South American fern, Pleopeltis minima (Polypodiaceae), and compared it to plant miRNA databases, allowing for the identification of miRNA families that are shared by all land plants, shared by all vascular plants (tracheophytes) or shared by euphyllophytes (ferns and seed plants) only. Using the recently described transcriptome of another fern, Lygodium japonicum, we also estimated the degree of conservation of fern miRNA targets in relation to other plant groups. Our results pinpoint the origin of several miRNA families in the land plant evolutionary tree with more precision and are a resource for future genomic and functional studies of fern miRNAs.

Fig 1. Phylogenetic position and habit of Pleopeltis minima.

(A) Simplified summary of phylogenetic relationships among major plant lineages relevant for the present study [3, 17, 24]. Note that some molecular phylogenies recover a clade composed of liverworts and mosses as sister to vascular plants [25, 26]. The fern (“monilophytes”) clade is the sister group to seed plants, gymnosperms and angiosperms. Within ferns, P. minima belongs to the order Polypodiales and is more closely related to Ceratopteris thalictroides, while Marsilea quadrifolia belongs to the order Salviniales and Lygodium japonicum to the order Schizaeales. (B) Habit of P. minima growing as epiphyte upon a tree trunk. (C) Abaxial side of leaf surface showing form and organization. (D) Portion of fertile leaf showing position of sori and scales. Scale bars: 5 mm.

Fig 2. Size distribution of P. minima sRNA sequences.

The graph displays the raw number of sRNA reads in the range of 18 to 25 nt. The percentage of reads in each size category in the range is indicated. A large peak at 21 nt and a smaller peak at 24 nt are noticeable.

Fig 3. Phylogenetic distribution of conserved fern miRNAs.

The grid indicates the presence (green) or absence (white) of miRNA families in liverworts (M. polymorpha and/or P. endiviifolia), mosses (P. patens), lycopods (S. moellendorffii), ferns (P. minima and M. quadrifolia) as well as gymnosperms, basal angiosperms, monocots and dicots. For gymnosperms we took into account miRNAs of P. abies [6, 41], C. lanceolata [38] and T. mairei [39] and species reported in [23]; for angiosperms we included miRNAs of species reported in [23] and in miRBase release 21.0 [31]. Pairs of miRNAs that can be considered from the same family are indicated by coloured dots. Note that miRNAs are not necessarily present in all members of a group. Among liverworts, miR530/1030, miR529 and miR536 are only found in M. polymorpha, while miR156, miR395 and miR535 are only found in P. endiviifolia [36, 42, 43]. MiR403 and miR529 are absent from most non-dicot angiosperms [44] and eudicots [32, 33], respectively.

Fig 4. Conserved targets of fern miRNAs.

Schematic representations of L. japonicum mRNAs predicted to be targeted by P. minima miRNAs. For comparison, homologous mRNAs and miRNAs from other land plants (A. thaliana or M. polymorpha) are also shown. Coding regions are shown as rectangles and UTR regions as thick black lines. Protein domains within coding regions are coloured. Regions predicted to be targeted by pmi-miRNAs are indicated by red stripes. Note that the positions of miRNA-targeted regions in mRNAs are conserved between L. japonicum and other plants. For pmi-miR408 the sequence for the homologous PPO mRNA from P. endiviifolia is not available and is not shown.

Fig 5. Trans-acting siRNA 3 targeting ARF transcripts in ferns.

(A) Schematic representations of two L. japonicum TAS3 RNAs (Locus 20755 and 39179). Predicted sites targeted by pmi-miR390 are indicated in red. The region predicted to generate a tasiRNA targeting a mRNA encoding a ARF transcription factor is indicated in blue. For comparison, a TAS3 transcript from A. thaliana (NR_143941.1) is also shown. (B) Schematic representation of a ARF mRNA (Locus 9356) from L. japonicum targeted by tasiRNA derived from TAS3 transcripts (blue stripe). The A. thaliana ARF3 mRNA (At2g33860) is shown for comparison. Conserved protein domains encoded by the ARF mRNAs are indicated.

Fig 6. Emergence of miRNAs during early land plant evolution.

The schematic cladogram shows the origin of ancient miRNA families during early land plant evolution. Only miRNA families that originated in early land plant evolution are shown, namely land plants (Embryophyta, node 1), mosses and vascular plants (node 2), vascular plants (Tracheophyta, node 3) and ferns and seed plants (Euphyllophyta, node 4). Note that, according to recent molecular phylogenies [25, 26], liverworts and mosses might belong together in a clade that is sister to vascular plants, in which case nodes 1 and 2 would be the same. Also note that five miRNA families seem to be restricted to euphyllophytes and that only miR1083 seems to have originated in the branch leading to vascular plants.