Evidence for the rapid expansion of microRNA-mediated regulation in early land plant evolution

BACKGROUND: MicroRNAs (miRNAs) are regulatory RNA molecules that are specified by their mode of action, the structure of primary transcripts, and their typical size of 20–24 nucleotides. Frequently, not only single miRNAs but whole families of closely related miRNAs have been found in animals and plants. Some families are widely conserved among different plant taxa. Hence, it is evident that these conserved miRNAs are of ancient origin and indicate essential functions that have been preserved over long evolutionary time scales. In contrast, other miRNAs seem to be species-specific and consequently must possess very distinct functions. Thus, the analysis of an early-branching species provides a window into the early evolution of fundamental regulatory processes in plants. RESULTS: Based on a combined experimental-computational approach, we report on the identification of 48 novel miRNAs and their putative targets in the moss Physcomitrella patens. From these, 18 miRNAs and two targets were verified in independent experiments. As a result of our study, the number of known miRNAs in Physcomitrella has been raised to 78. Functional assignments to mRNAs targeted by these miRNAs revealed a bias towards genes that are involved in regulation, cell wall biosynthesis and defense. Eight miRNAs were detected with different expression in protonema and gametophore tissue. The miRNAs 1–50 and 2–51 are located on a shared precursor that are separated by only one nucleotide and become processed in a tissue-specific way. CONCLUSION: Our data provide evidence for a surprisingly diverse and complex miRNA population in Physcomitrella. Thus, the number and function of miRNAs must have significantly expanded during the evolution of early land plants. As we have described here within, the coupled maturation of two miRNAs from a shared precursor has not been previously identified in plants

Accumulation dynamics of transcripts and proteins of cold-responsive genes in fragaria vesca genotypes of differing cold tolerance

Identifying and characterizing cold responsive genes in Fragaria vesca associated with or responsible for low temperature tolerance is a vital part of strawberry cultivar development. In this study we have investigated the transcript levels of eight genes, two dehydrin genes, three putative ABA-regulated genes, two cold–inducible CBF genes and the alcohol dehydrogenase gene, extracted from leaf and crown tissues of three F. vesca genotypes that vary in cold tolerance. Transcript levels of the CBF/DREB1 transcription factor FvCBF1E exhibited stronger cold up-regulation in comparison to FvCBF1B.1 in all genotypes. Transcripts of FvADH were highly up-regulated in both crown and leaf tissues from all three genotypes. In the ‘ALTA’ genotype, FvADH transcripts were significantly higher in leaf than crown tissues and more than 10 to 20-fold greater than in the less cold-tolerant ‘NCGR1363’ and ‘FDP817’ genotypes. FvGEM, containing the conserved ABRE promoter element, transcript was found to be cold-regulated in crowns. Direct comparison of the kinetics of transcript and protein accumulation of dehydrins was scrutinized. In all genotypes and organs, the changes of XERO2 transcript levels generally preceded protein changes, while levels of COR47 protein accumulation preceded the increases in COR47 RNA in ‘ALTA’ crowns.publishedVersio

Expression of artificial microRNAs in Physcomitrella patens

MicroRNAs (miRNAs) are ~21nt long small RNAs transcribed from endogenous MIR genes which form precursor RNAs with a characteristic hairpin structure. MiRNAs control the expression of cognate target genes by binding to reverse complementary sequences resulting in cleavage or translational inhibition of the target RNA. Artificial miRNAs (amiRNAs) can be generated by exchanging the miRNA/miRNA* sequence of endogenous MIR precursor genes, while maintaining the general pattern of matches and mismatches in the foldback. Thus, for functional gene analysis amiRNAs can be designed to target any gene of interest

Physcomitrella patens small RNA pathways

Small, non-coding RNAs (sRNAs) are a distinct class of regulatory RNAs in plants and animals controlling a variety of biological processes. Given the great impact of sRNAs in biology, recent studies in model seed plant species, particu-larly in A. thaliana, focused on the identification, biogenesis and functional analysis of sRNAs. In seed plants, several classes of sRNAs with specific sizes and dedicated functions have evolved through a series of pathways, namely microR-NAs (miRNAs), repeat-associated small interfering RNAs (ra-siRNAs), natural antisense transcript-derived small interfering RNAs (nat-siRNAs), and trans-acting small interfering RNAs (ta-siRNAs). In the last years, the analysis of plant sRNA pathways was extended to the bryophyte P. patens, a non-flowering, non-vascular ancient land plant, that diverged from the lineage of seed plants approxi-mately 450 million years ago. Based on a number of characteristic features and its phylogenetic key position in land plant evolution P. patens emerged as a plant model species to address basic as well as applied topics in plant biology. The analysis of P. patens sRNA pathways was recently advanced by the deep sequencing of sRNA libraries, the release of the P. patens genome that allowed the mapping of sRNA producing loci, and first molecular analyses of P. patens mutants with targeted disruption of genes encoding essential components of endogenous sRNA pathways. Even though the major sRNA pathways are evolutionarily conserved in P. patens there are particular differences in the functional components of sRNA pathways and the biological function of sRNAs. These include a specific amplification of initial miRNA and ta-siRNA signals by the generation of transitive siRNAs, deviating functions and specificities of DICER-LIKE proteins and an epigenetic gene silencing pathway that is triggered by miRNAs. Further, the conservation of miRNA biogenesis in P. patens was used to establish specific gene silencing by the expression of artificial miRNAs suited for functional gene analysis by reverse genetics approaches. These findings underline that P. patens serves as a valuable model system to study the evolution, diversity, and function of plant sRNAs. Here we summarise the current knowledge on different sRNA biogenesis pathways, their biological relevance and the expres-sion of artificial miRNAs in P. patens

Remobilization and copy number increase.

<p>(A) Inverse PCR amplification of T7-<i>neo</i> insertion sites of the colonies on higher level of G418 (see Materials and Methods). C, negative control using the genomic DNA of the yeast strain. (B) T7-<i>neo</i> insertion sites in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064135#pone.0064135.s004" target="_blank">Table S4</a> mapped on yeast schematic chromosomes. Short vertical bars, insertion sites with the red bar representing the inferred first insertion on chromosomes.</p

Excision assay of T7 derived constructs in yeast.

<p>(A) plates of medium lacking adenine in the presence (bottom row) and absence of transposase sources (top row), cells in each sector on a plate were from a separate colony incubated on medium lacking histidine and uracil for 14 days. (B) PCR amplification of the donor sites in the <i>ade2</i> revertants. C, plasmid control; (C) Sequences of the donor sites in the <i>ade2</i> revertants. Red letters, target site dinucleotide “TA”. Dashes, empty space. The donor sites containing TEs are shown on top of the donor sites containing footprints. (D) Reversion frequency for the constructs T7, T7-<i>neo</i>, T7-<i>gfp</i>, T7-<i>neo</i>-<i>gfp</i>. The reversion frequency (left y-axis) is plotted against the length of the cargo cassettes (right y-axis). Error bars, standard error of the mean for three independent replicates. Plates were incubated on transposase induction medium for nine days.</p

Insertion of T7-<i>neo</i>.

<p>(A) schematic experimental procedure to select for genomic insertion of T7-<i>neo</i> (see Materials and Methods); (B) selection of G418 resistant colonies in the presence (right) and absence of (left) transposase sources; (C) PCR amplification of <i>ade</i>2 gene coding sequence; N, negative control using DG2523 genomic DNA; P, positive control using plasmid pT7-<i>neo</i>. (D) growth of G418 resistant colonies on complete synthetic medium with (bottom) or without (top) uracil. (E) Genomic DNA blot analysis. P, plasmid (pT7-<i>neo</i>) control; N, negative control of yeast genomic DNA not containing T7-<i>neo</i> fragment. Both images were from the same blot but hybridized with different probes. (F) Inverse PCR amplification of insertion sites in G418 resistant colonies. C, control using DG2523 yeast genomic DNA. Plates were incubated on transposase induction medium for two weeks.</p

A Rice <i>Stowaway</i> MITE for Gene Transfer in Yeast

<div><p>Miniature inverted repeat transposable elements (MITEs) lack protein coding capacity and often share very limited sequence similarity with potential autonomous elements. Their capability of efficient transposition and dramatic amplification led to the proposition that MITEs are an untapped rich source of materials for transposable element (TE) based genetic tools. To test the concept of using MITE sequence in gene transfer, a rice <i>Stowaway</i> MITE previously shown to excise efficiently in yeast was engineered to carry cargo genes (<i>neo</i> and <i>gfp</i>) for delivery into the budding yeast genome. Efficient excision of the cargo gene cassettes was observed even though the excision frequency generally decreases with the increase of the cargo sizes. Excised elements insert into new genomic loci efficiently, with about 65% of the obtained insertion sites located in genes. Elements at the primary insertion sites can be remobilized, frequently resulting in copy number increase of the element. Surprisingly, the orientation of a cargo gene (<i>neo</i>) on a construct bearing dual reporter genes (<i>gfp</i> and <i>neo</i>) was found to have a dramatic effect on transposition frequency. These results demonstrated the concept that MITE sequences can be useful in engineering genetic tools to deliver cargo genes into eukaryotic genomes.</p></div

Effect of the orientation of <i>neo</i> gene on transposition efficiency.

<p>(A) schematic structure of the T7-<i>gfp</i>-<i>oen</i> cargo gene cassette. Legends are the same as those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064135#pone-0064135-g001" target="_blank">Fig. 1</a>. (B) Relative <i>ade</i>2 reversion efficiency (left) and cargo gene cassette insertion frequency (right) of the three constructs. The mean values of both reversion frequency and insertion frequency for T7-<i>gfp</i> were arbitrarily designated as 100%. Error bar, standard error of the mean for six independent replicates.</p