The liverwort Pellia endiviifolia shares microtranscriptomic traits that are common to green algae and land plants

Abstract

Liverworts are the most basal group of extant land plants. Nonetheless, the molecular biology of liverworts is poorly understood. Gene expression has been studied in only one species, Marchantia polymorpha. In particular, no microRNA (miRNA) sequences from liverworts have been reported. Here, Illumina-based next-generation sequencing was employed to identify small RNAs, and analyze the transcriptome and the degradome of Pellia endiviifolia. Three hundred and eleven conserved miRNA plant families were identified, and 42 new liverwort-specific miRNAs were discovered. The RNA degradome analysis revealed that target mRNAs of only three miRNAs (miR160, miR166, and miR408) have been conserved between liverworts and other land plants. New targets were identified for the remaining conserved miRNAs. Moreover, the analysis of the degradome permitted the identification of targets for 13 novel liverwort-specific miRNAs. Interestingly, three of the liverwort microRNAs show high similarity to previously reported miRNAs from Chlamydomonas reinhardtii. This is the first observation of miRNAs that exist both in a representative alga and in the liverwort P. endiviifolia but are not present in land plants. The results of the analysis of the P. endivifolia microtranscriptome support the conclusions of previous studies that placed liverworts at the root of the land plant evolutionary tree of life.

Keywords: Pellia endiviifolia; basal lineage; land plants; liverwort; miRNA precursors; miRNA targets; microRNA (miRNA) genes; microtranscriptome.

Figure 1

Venn diagram showing the identification of Pellia endiviifolia conservative microRNA (miRNA) families within land plants according to miRBase [http://www.mirbase.org/].

Figure 2

Selected conservative microRNAs (miRNAs) in the liverwort Pellia endiviifolia as detected by northern hybridization. Above the blots, the name of each miRNA is provided along with the length of the dominating cDNA fragment in a given cluster and the mean counts (k, kilo) from all of the deep-sequencing results for all family members (up to two mismatches in the overlapping region to the probe sequence were considered; see Supporting Information Table S2). (a) pen-miRNA 408. The graph on the right presents the distribution of reads within the miR408 cluster. The read counts represent a single next-generation sequencing (NGS) experiment. (b) miRNA166, miR168, and miR319. The left side of the northern blots shows the RNA decade marker depicting 30-nucleotide (nt)- and 20-nt-long RNAs.

Figure 3

Forty-two novel microRNAs (miRNAs) in the liverwort Pellia endiviifolia as detected by northern hybridization. Above the blots, the length of the dominating cDNA fragment in a given cluster and the mean counts (k, kilo) from all of the deep-sequencing results are provided. (a) pen-miRNA8165. The graph on the right shows the distribution of the reads within the pen-miR8165 cluster. The read counts represent a single next-generation sequencing (NGS) experiment. (b) Additional novel pen-miRNAs. The left side of the northern blots shows the RNA decade marker depicting a 20-nucleotide (nt)-long RNA. The numbers above the blots represent the numerical part of the novel miRNA ID. Open circle, pen-miR8156a, pen-miR8156b, pen-miR8156c and pen-miR8156d form one miRNA family and differ from each other by 1 nt; closed circles, sRNA homologs that were found in the Chlamydomonas reinhardtii NGS data with one and two mismatches in the overlapping regions. Open square, pen-miR408-5p*. miR408-5p* was originally identified as a novel miRNA and then identified as miR* in pre-miRNA408-5p.

Figure 4

Selected Pellia endiviifolia microRNAs (miRNAs) that were homologous to Chlamydomonas reinhardtii miRNAs as detected by northern hybridization. The left side of the northern blots shows the RNA decade marker. Above the blots, the name of each miRNA is provided along with its length and mean counts (k, kilo). nt, nucleotide.

Figure 5

miR8162-5p and miR8162-3p derive from the same transcript, generating a stem-loop structure. The left panel shows the stem-loop structure of the pen-miR8162 precursor. Both of the microRNA (miRNA) species are marked in green because both are present at similar levels, and putative targets were found for both. The right panel shows northern hybridization for both miRNA species. nt, nucleotide; k, kilo.

Figure 6

Target plots (T-plots) for Pellia endiviifolia microRNA (miRNA) targets. T-plots (German et al., 2008) are shown for known P. endiviifolia miRNA targets that are (a) conserved across the land plants and (b) new, validated using degradome data and corresponding to the targets that are presented in Table5. Each T-plot is accompanied by a duplex of miRNA and its target mRNA. For miR408 targeting homologous mRNAs and recognizing/cleaving the same sequence, only one example of each group of targets is shown (four T-plots). TP1M is the normalized abundance based on the formula TP1M = (raw abundance/library size) × 10⁶. Additionally, the normalized TP1M value was divided by the number of targets to which it perfectly matched. The red line indicates the position of the predicted cleavage site as confirmed by an aligned degradome tag.

Figure 7

Target plots (T-plots) of validated target mRNAs for novel Pellia endiviifolia miRNAs using degradome data corresponding to the targets that are presented in Table6. The first T-plot shows the target coding sequences for three new microRNAs (miRNAs) representing a new miRNA family, pen-miR8156. The next two plots show the predicted slicing site for miR8158 and miR8166, representing the putative transcription factor mRNAs. TP1M abundance is normalized based on the formula TP1M = (raw abundance/library size) × 10⁶. Additionally, the normalized TP1M value was divided by the number of targets to which it perfectly matched. Each T-plot is accompanied by a duplex of miRNA and its target mRNA. The nucleotides that are shown in red represent nucleotide mismatches within the miR8156 family. bp, base pairs.