Expression-Based Functional Investigation of the Organ-Specific MicroRNAs in <em>Arabidopsis</em>

<div><p>MicroRNAs (miRNAs) play a pivotal role in plant development. The expression patterns of the miRNA genes significantly influence their regulatory activities. By utilizing small RNA (sRNA) high-throughput sequencing (HTS) data, the miRNA expression patterns were investigated in four organs (flowers, leaves, roots and seedlings) of <em>Arabidopsis</em>. Based on a set of criteria, dozens of organ-specific miRNAs were discovered. A dominant portion of the organ-specific miRNAs identified from the ARGONAUTE 4-enriched sRNA HTS libraries were highly expressed in flowers. Additionally, the expression of the precursors of the organ-specific miRNAs was analyzed. Degradome sequencing data-based approach was employed to identify the targets of the organ-specific miRNAs. The miRNA–target interactions were used for network construction. Subnetwork analysis unraveled some novel regulatory cascades, such as the feedback regulation mediated by miR161, the potential self-regulation of the genes <em>miR172</em>, <em>miR396</em>, <em>miR398</em> and <em>miR860</em>, and the miR863-guided cleavage of the <em>SERRATE</em> transcript. Our bioinformatics survey expanded the organ-specific miRNA–target list in <em>Arabidopsis</em>, and could deepen the biological view of the miRNA expression and their regulatory roles.</p> </div

Genome-Wide Identification of Reverse Complementary microRNA Genes in Plants

<div><p>MicroRNAs (miRNAs) are ∼21-nucleotide small RNAs (sRNAs) with essential regulatory roles in plants. They are generated from stem-loop-structured precursors through two sequential Dicer-like 1 (DCL1)-mediated cleavages. To date, hundreds of plant miRNAs have been uncovered. However, the question, whether the sequences reverse complementary (RC) to the miRNA precursors could form hairpin-like structures and produce sRNA duplexes similar to the miRNA/miRNA* pairs has not been solved yet. Here, we interrogated this possibility in 16 plant species based on sRNA high-throughput sequencing data and secondary structure prediction. A total of 59 RC sequences with great potential to form stem-loop structures and generate miRNA/miRNA*-like duplexes were identified in ten plants, which were named as RC-miRNA precursors. Unlike the canonical miRNAs, only a few cleavage targets of the RC-miRNAs were identified in <em>Arabidopsis</em> (<em>Arabidopsis thaliana</em>) and rice (<em>Oryza sativa</em>), and none in Soybean (<em>Glycine max</em>) based on degradome data. Surprisingly, the genomic regions surrounding some of the RC-miRNA target recognition sites were observed to be specifically methylated in both <em>Arabidopsis</em> and rice. Taken together, we reported a new class of miRNAs, called RC-miRNAs, which were generated from the antisense strands of the miRNA precursors. Based on the results, we speculated that the mature RC-miRNAs might have subtle regulatory activity through target cleavages, but might possess short interfering RNA-like activity by guiding sequence-specific DNA methylation.</p> </div

Site-specific DNA methylation mediated by the highly accumulated reverse complementary microRNAs (RC-miRNAs) in <i>Arabidopsis</i> and rice.

<p>(A) and (B) RC-ath-miR782-2-mediated DNA methylation on the region encoding <i>AT1G04160</i> and <i>AT2G33240</i>. (C) RC-ath-miR847-2-mediated DNA methylation on the region encoding <i>AT3G07610</i>. (D) RC-oas-miR2118q-mediated DNA methylation on the region encoding <i>LOC_Os03g22570</i>. (E) RC-oas-miR169p/q-mediated DNA methylation on the region encoding <i>LOC_Os03g29760</i>. (F) RC-oas-miR1846e-mediated DNA methylation on the region encoding <i>LOC_Os07g32530</i>. The genomic positions of the target binding sites of the RC-miRNAs are shown on the top right corners of the screenshots of the public available epigenome browsers of <i>Arabidopsis</i> and rice <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046991#pone.0046991-He1" target="_blank">[29]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046991#pone.0046991-Zhang2" target="_blank">[30]</a>.</p

Degradome sequencing data-based identification of the targets of the highly accumulated reverse complementary microRNAs (RC-miRNAs) in <i>Arabidopsis</i> and rice.

<p>For all the sub-figures (A to F), the left panels depict the degradome signals all along the target transcripts, and the other panels provide detailed views of the cleavage signals within the regions surrounding the target recognition sites (denoted by gray horizontal lines). The transcript IDs are shown in the left panels, and the RC-miRNA names are listed in the other panels. The <i>x</i> axes measure the positions of the signals along the transcripts, and the <i>y</i> axes measure the signal intensities based on normalized counts (in RPM, reads per million), allowing cross-library comparison. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046991#pone.0046991.s008" target="_blank">Table S2</a> for the degradome data sets used in this analysis.</p

Certain intriguing subnetworks mediated by organ-specific microRNAs.

<p>(<b>A</b>) ath-miR161.2-mediated subnetwork involving <i>PPR</i> (<i>pentatricopeptide repeat</i>) genes and <i>trans</i>-acting small interfering RNA (ta-siRNA)-generating (<i>TAS</i>) genes. (<b>B</b>) miR172-involved self-regulatory network. (<b>C</b>) miR396-involved self-regulatory network. (<b>D</b>) miR398-involved self-regulatory network. (<b>E</b>) miR860-involved self-regulatory network. (<b>F</b>) ath-miR863-3p-mediated regulation of <i>SERRATE</i>. For (<b>A</b>) to (<b>F</b>), the degradome-based evidences for the microRNA-mediated target cleavages are shown by the target plots (t-plots). The target transcripts and the microRNAs are listed on the top of each plot. The <i>x</i> axes measure the positions of the target binding regions (indicated by blue bars) on the target transcripts. The <i>y</i> axes measure the intensity (in RPM, reads per million) of the cleavage signals based on the degradome sequencing data. The degradome signals belonging to GSM278370 and AxSRP which were prepared from the <i>Arabidopsis</i> seedlings were represented by black dots, and those from the libraries prepared from the flowers were represented by gray ones (see detailed library information in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050870#s4" target="_blank">Materials and Methods</a></b>). The prominent cleavage sites are denoted by red dashed lines.</p

List of the organ-specific microRNAs in <i>Arabidopsis</i>.

1<p>Organ-specific microRNAs (miRNAs) identified from the WT-related library group.</p>2<p>Organ-specific miRNAs identified from the AGO1-related library group.</p>3<p>Organ-specific miRNAs identified from the AGO4-related library group.</p>4<p>Organ-specific miRNAs in both the WT- and the AGO1-related groups.</p>5<p>Organ-specific miRNAs in both the WT- and the AGO4-related groups.</p>6<p>Organ-specific miRNAs in both the AGO1- and the AGO4-related groups.</p>7<p>Organ-specific miRNAs in the WT-, the AGO1- and the AGO4-related groups.</p>8<p>Flower-specific miRNAs.</p>9<p>Leaf-specific miRNAs.</p>10<p>Root-specific miRNAs.</p>11<p>Seedling-specific miRNAs.</p><p>Please refer to <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050870#s4" target="_blank">Materials and Methods</a></b> for the definition of the WT-, the AGO1- and the AGO4-related library groups.</p><p>See <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050870#s4" target="_blank">Materials and Methods</a></b> for the identification of the organ-specific miRNAs.</p

Separation of abietane-type diterpenoids from <i>Clerodendrum kaichianum</i> Hsu by high-speed counter-current chromatography using stepwise elution

<p>High-speed counter-current chromatography (HSCCC) was successfully used for the separation of abietane-type diterpenoids from the medicinal plant <i>C. kaichianum</i>, which were not separated in our previous study using preparative HPLC. The HSCCC separation employed the lower phases of <i>n</i>-hexane–ethyl acetate–methanol–water (HEMW) 4:5:4:5 and HEMW 4:5:5:4 as the mobile phase for stepwise elution while the upper phase of HEMW 4:5:4:5 was used as the stationary phase. HSCCC separation yielded 90.5 mg of compound <b>1</b>(kaichianone A), 137.7 mg of compound <b>2</b> (kaichianone B), 125.0 mg of compound <b>3</b> (teuvincenone E), and 227.6 mg of compound <b>4</b> (taxusabietane A) with purities of 95.3%, 97.2%, 97.8%, and 98.6%, respectively, as determined by HPLC. Compounds <b>1</b>–<b>2</b> are two new abietane-type diterpenoids while Compounds <b>3</b>–<b>4</b> are known abietane-type diterpenoids, analyzed by ESIMS and NMR data. The results demonstrated that HSCCC can be an excellent alternative for other separation methods. The two new compounds showed significant cytotoxicity against ileocecal carcinoma HCT-8 and breast adenocarcinoma MCF-7 cells.</p> <p>High-speed counter-current chromatography (HSCCC) was successfully used for the separation of abietane-type diterpenoids from the medicinal plant <i>C. kaichianum</i>, which were not separated in our previous study using preparative HPLC. </p

Dependence of RC-osa-miR1857 on DCL1, but not DCL3 and RDR2, and its enrichment in Argonaute 1.

<p>Secondary structure and the accumulation levels of the mature RC-miRNA (indicated by red color) and the RC-miRNA* (blue color) are shown on the top of the figure. The table at the bottom shows the accumulation levels of RC-osa-miR1857 in various biological samples, such as wild type seedling, <i>dcl1</i> mutant, <i>DCL1</i> RNAi (RNA interference) transgenic lines, <i>dcl3</i> mutant, <i>rdr2</i> mutant, and the sequencing data from Argonaute 1 (AGO1)-enriched small RNA population. Please note, the expression data (normalized in RPM, reads per million) was more comparable for the high-throughput sequencing data sets belonging to the same group (indicated by the same background color).</p

Organ-specific validation of the microRNA targets based on degradome sequencing data.

<p>For (<b>A</b>) to (<b>L</b>), the target transcripts and the microRNA regulators are listed on the top of each target plot (t-plot). The <i>x</i> axes measure the positions of the target binding regions (indicated by gray bars) on the target transcripts. The <i>y</i> axes measure the intensity (in RPM, reads per million) of the cleavage signals based on the degradome data. The degradome signals belonging to GSM278370 and AxSRP which were prepared from the <i>Arabidopsis</i> seedlings were represented by black dots, and those from the libraries prepared from the flowers were represented by gray ones (see detailed library information in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050870#s4" target="_blank">Materials and Methods</a></b>). The prominent cleavage sites are denoted by dashed lines. (<b>M</b>) Expression levels of the mature microRNAs listed in (<b>A</b>) to (<b>L</b>) in the seedlings and the flowers. Based on the small RNA high-throughput sequencing data, the microRNAs specifically expressed in the seedlings or the flowers (either in the WT-related library group, or in the AGO1-related library group, or in the AGO4-related library group; see detailed information in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050870#s4" target="_blank">Materials and Methods</a></b>) were indicated by black “S” or gray “F” respectively.</p

Organ-specific microRNA-mediated regulatory networks.

<p>(<b>A</b>) Network mediated by flower-specific microRNAs. (<b>B</b>) Network mediated by leaf-specific microRNAs. (<b>C</b>) Network mediated by root-specific microRNAs. (<b>D</b>) Network mediated by seedling-specific microRNAs. (<b>E</b>) Network mediated by the microRNAs specifically expressed in roots and seedlings. (<b>F</b>) Network mediated by the microRNAs specifically expressed in leaves and roots. (<b>G</b>) Network mediated by the microRNAs specifically expressed in leaves and seedlings. (<b>H</b>) Network mediated by the microRNAs specifically expressed in flowers and roots. (<b>I</b>) Network mediated by the microRNAs specifically expressed in leaves, roots, and seedlings. (<b>J</b>) Network mediated by the microRNAs specifically expressed in flowers and seedlings. (<b>K</b>) Network mediated by the microRNAs specifically expressed in flowers and leaves. All the networks were constructed based on the validated microRNA target list by using Cytoscape.²⁹ The different color bar combinations indicate the specific expression patterns of the microRNAs involved in each network. See the bottom right for the meanings of the color bars.</p