Ambient temperature regulates the expression of a small set of sRNAs influencing plant development through NF-YA2 and YUC2.

Plants substantially alter their developmental programme upon changes in the ambient temperature. The 21-24 nt small RNAs (sRNAs) are important gene expression regulators, which play a major role in development and adaptation. However, little is known about how the different sRNA classes respond to changes in the ambient temperature. We profiled the sRNA populations in four different tissues of Arabidopsis thaliana plants grown at 15°C, 21°C, and 27°C. We found that only a small fraction (0.6%) of the sRNA loci are ambient temperature-controlled. We identified thermoresponsive microRNAs and identified their target genes using degradome libraries. We verified that the target of the thermoregulated miR169, NF-YA2, is also ambient temperature-regulated. NF-YA2, as the component of the conserved transcriptional regulator NF-Y complex, binds the promoter of the flowering time regulator FT and the auxin biosynthesis gene YUC2. Other differentially expressed loci include thermoresponsive phased siRNA loci that target various auxin pathway genes and tRNA fragments. Furthermore, a temperature-dependent 24-nt heterochromatic siRNA locus in the promoter of YUC2 may contribute to the epigenetic regulation of auxin homeostasis. This holistic approach facilitated a better understanding of the role of different sRNA classes in ambient temperature adaptation of plants.

Identification of Nicotiana benthamiana microRNAs and their targets using high throughput sequencing and degradome analysis.

BACKGROUND: Nicotiana benthamiana is a widely used model plant species for research on plant-pathogen interactions as well as other areas of plant science. It can be easily transformed or agroinfiltrated, therefore it is commonly used in studies requiring protein localization, interaction, or plant-based systems for protein expression and purification. To discover and characterize the miRNAs and their cleaved target mRNAs in N. benthamiana, we sequenced small RNA transcriptomes and degradomes of two N. benthamiana accessions and validated them by Northern blots. RESULTS: We used a comprehensive molecular approach to detect and to experimentally validate N. benthamiana miRNAs and their target mRNAs from various tissues. We identified 40 conserved miRNA families and 18 novel microRNA candidates and validated their target mRNAs with a genomic scale approach. The accumulation of thirteen novel miRNAs was confirmed by Northern blot analysis. The conserved and novel miRNA targets were found to be involved in various biological processes including transcription, RNA binding, DNA modification, signal transduction, stress response and metabolic process. Among the novel miRNA targets we found the mRNA of REPRESSOR OF SILENCING (ROS1). Regulation of ROS1 by a miRNA provides a new regulatory layer to reinforce transcriptional gene silencing by a post-transcriptional repression of ROS1 activity. CONCLUSIONS: The identified conserved and novel miRNAs along with their target mRNAs also provides a tissue specific atlas of known and new miRNA expression and their cleaved target mRNAs of N. benthamiana. Thus this study will serve as a valuable resource to the plant research community that will be beneficial well into the future.

Expansion of Capsicum annuum fruit is linked to dynamic tissue-specific differential expression of miRNA and siRNA profiles.

Small regulatory RNAs, such as microRNAs (miRNAs) and small interfering RNAs (siRNAs) have emerged as important transcriptional and post-transcriptional regulators controlling a wide variety of physiological processes including fruit development. Data are, however, limited for their potential roles in developmental processes determining economically important traits of crops. The current study aimed to discover and characterize differentially expressed miRNAs and siRNAs in sweet pepper (Capsicum annuum) during fruit expansion. High-throughput sequencing was employed to determine the small regulatory RNA expression profiles in various fruit tissues, such as placenta, seed, and flesh at 28 and 40 days after anthesis. Comparative differential expression analyses of conserved, already described and our newly predicted pepper-specific miRNAs revealed that fruit expansion is accompanied by an increasing level of miRNA-mediated regulation of gene expression. Accordingly, ARGONAUTE1 protein, the primary executor of miRNA-mediated regulation, continuously accumulated to an extremely high level in the flesh. We also identified numerous pepper-specific, heterochromatin-associated 24-nt siRNAs (hetsiRNAs) which were extremely abundant in the seeds, as well as 21-nt and 24-nt phased siRNAs (phasiRNAs) that were expressed mainly in the placenta and the seeds. This work provides comprehensive tissue-specific miRNA and siRNA expression landscape for a developing pepper fruit. We identified several novel, abundantly expressing tissue- and pepper-specific small regulatory RNA species. Our data show that fruit expansion is associated with extensive changes in sRNA abundance, raising the possibility that manipulation of sRNA pathways may be employed to improve the quality and quantity of the pepper fruit.

Correction: Expansion of Capsicum annum fruit is linked to dynamic tissue-specific differential expression of miRNA and siRNA profiles.

[This corrects the article DOI: 10.1371/journal.pone.0200207.].

Identification of ARGONAUTE/Small RNA Cleavage Sites by Degradome Sequencing.

The method described here enables the high-throughput identification of cleaved mRNA targets of ARGONAUTE/small RNA complexes. The protocol is based on a modified 5'-rapid amplification of cDNA ends combined with deep sequencing of 3' cleavage products of mRNAs. Poly(A) RNA is purified from the tissue of interest which is followed by a 5'-RNA adapter ligation. The ligated products are then reverse transcribed, amplified, and digested with MmeI. After gel separation, a 3' double-stranded DNA adapter is ligated to the fragments, which are then amplified and index labeled for the high-throughput sequencing of pooled degradome libraries. Sequencing datasets from pooled libraries can be analyzed with different bioinformatic approaches.