Summary: Micro‐RNAs (miRNAs) are one class of endogenous tiny RNAs that play important regulatory roles in plant development and responses to external stimuli. To date, miRNAs have been cloned from higher plants such as Arabidopsis, rice and pumpkin, and there is limited information on their identity in lower plants including Bryophytes. Bryophytes are among the oldest groups of land plants among the earth's flora, and are important for our understanding of the transition to life on land. To identify miRNAs that might have played a role early in land plant evolution, we constructed a library of small RNAs from the juvenile gametophyte (protonema) of the moss Physcomitrella patens. Sequence analysis revealed five higher plant miRNA homologues, including three members of the miR319 family, previously shown to be involved in the regulation of leaf morphogenesis, and miR156, which has been suggested to regulate several members of the SQUAMOSA PROMOTER BINDING‐LIKE (SPL) family in Arabidopsis. We have cloned PpSBP3, a moss SPL homologue that contains an miR156 complementary site, and demonstrated that its mRNA is cleaved within that site suggesting that it is an miR156 target in moss. Six additional candidate moss miRNAs were identified and shown to be expressed in the gametophyte, some of which were developmentally regulated or upregulated by auxin. Our observations suggest that miRNAs play important regulatory roles in mosses.

Being composed of several whorls of distinct floral organs, the flower is one of the most complex organs in the plant. As such, the formation and maintenance of boundaries that separate the meristem from the floral organ primordium and adjacent organs are critical for its normal development. In Arabidopsis, the miR164-regulated NAM genes play key roles in floral-boundary specification. By contrast, much less is known about floral-boundary establishment in the model crop tomato. It was found that the miR164-regulated NAM gene GOBLET is expressed in the floral meristem–organ boundaries and its loss-of-function mutant produces flowers with fused organs, indicating its requirement for tomato floral-boundary formation. It was found here that sly-miR164 targets the transcripts of three additional uncharacterized NAM genes in developing flowers. It is shown that, after floral-boundary initiation, the NAM gene Solyc03g115850 (SlNAM2) is expressed as stripes that mark the boundaries between sepals and between different floral whorls. Furthermore, ectopic accumulation of SlNAM2-encoding transcripts caused various growth-suppression and extraorgan phenotypes typically observed in plants over-expressing known boundary genes. Flower-specific silencing of sly-miR164-targeted NAM genes (AP1>>MIR164) caused defects in the separation of sepals and floral whorls indicating abnormal boundary specification. However, supplementing these NAM-deficient flowers with miR164-resistant SlNAM2 suppressed their fusion phenotypes and completely restored floral boundaries. Together, our results strongly suggest that SlNAM2 participates in the establishment of tomato flower whorl and sepal boundaries.

In plants, cytosine methylation, an epigenetic mark critical for transposon silencing, is maintained over generations by key enzymes that directly methylate DNA and is facilitated by chromatin remodelers, like DECREASE IN DNA METHYLATION 1 (DDM1). Short-interfering RNAs (siRNAs) also mediate transposon DNA methylation through a process called RNA-directed DNA methylation (RdDM). In tomato (Solanum lycopersicum), siRNAs are primarily mapped to gene-rich chromosome arms, and not to pericentromeric regions as in Arabidopsis thaliana. Tomato encodes two DDM1 genes. To better understand their functions and interaction with the RdDM pathway, we targeted the corresponding genes via the CRISPR/Cas9 technology, resulting in the isolation of Slddm1a and Slddm1b knockout mutants. Unlike the single mutants, Slddm1a Slddm1b double mutant plants display pleiotropic vegetative and reproductive phenotypes, associated with severe hypomethylation of the heterochromatic transposons in both the CG and CHG methylation contexts. The methylation in the CHH context increased for some heterochromatic transposons and conversely decreased for others localised in euchromatin. We found that the number of heterochromatin-associated siRNAs, including RdDM-specific small RNAs, increased significantly, likely limiting the transcriptional reactivation of transposons in Slddm1a Slddm1b. Taken together, we propose that the global production of siRNAs and the CHH methylation mediated by the RdDM pathway are restricted to chromosome arms in tomato. Our data suggest that both pathways are greatly enhanced in heterochromatin when DDM1 functions are lost, at the expense of silencing mechanisms normally occurring in euchromatin.

Summary: Trans‐acting small interfering RNAs (tasiRNAs) are a class of higher‐plant endogenous siRNAs that, like miRNAs, direct the cleavage of non‐identical transcripts. tasiRNAs derive from non‐coding transcripts (TAS) that are converted into dsRNA by a RNA‐dependent RNA polymerase (RDR6), following their initial miRNA‐guided cleavage. The dsRNA is then processed by a dicer‐like enzyme 4 into phased 21‐nucleotide siRNAs. To date, tasiRNAs have been identified only in Arabidopsis, and their identity and function in other land plants are unknown. Here, a set of endogenous small RNAs that correspond in a phased manner to a non‐coding transcript (contig13502) were identified in the moss Pyscomitrella patens. Northern analysis suggests that contig13502‐derived small RNAs are expressed in the juvenile gametophyte. In addition, miR390‐guided cleavage of contig13502 at two sites flanking the small RNAs cluster was validated by 5′ RACE. These cleavages are predicted to provide defined termini for the production of phased siRNAs. To elucidate the biogenesis of identified siRNAs, we cloned and generated knock‐out mutants for an RDR6 moss homologue (PpRDR6). These mutants exhibited an accelerated transition from juvenile to mature gametophyte. In addition, RNA blots demonstrated that they lacked contig13502‐derived siRNAs, suggesting that PpRDR6 is required for siRNA biogenesis. A target gene, which showed homology to an AP2/EREBP transcription factor, for one phased siRNA, was validated, corroborating its identity as a trans‐acting siRNA. Taken together, our data indicate that contig13502 is a novel TAS locus and suggest a role for derived tasiRNAs in the regulation of gene expression in moss.