The hot pepper (Capsicum annuum) microRNA transcriptome reveals novel and conserved targets: a foundation for understanding MicroRNA functional roles in hot pepper.

MicroRNAs (miRNAs) are a class of non-coding RNAs approximately 21 nt in length which play important roles in regulating gene expression in plants. Although many miRNA studies have focused on a few model plants, miRNAs and their target genes remain largely unknown in hot pepper (Capsicum annuum), one of the most important crops cultivated worldwide. Here, we employed high-throughput sequencing technology to identify miRNAs in pepper extensively from 10 different libraries, including leaf, stem, root, flower, and six developmental stage fruits. Based on a bioinformatics pipeline, we successfully identified 29 and 35 families of conserved and novel miRNAs, respectively. Northern blot analysis was used to validate further the expression of representative miRNAs and to analyze their tissue-specific or developmental stage-specific expression patterns. Moreover, we computationally predicted miRNA targets, many of which were experimentally confirmed using 5' rapid amplification of cDNA ends analysis. One of the validated novel targets of miR-396 was a domain rearranged methyltransferase, the major de novo methylation enzyme, involved in RNA-directed DNA methylation in plants. This work provides the first reliable draft of the pepper miRNA transcriptome. It offers an expanded picture of pepper miRNAs in relation to other plants, providing a basis for understanding the functional roles of miRNAs in pepper.

Hwang DG ，Park JH ，Lim JY ，Kim D ，Choi Y ，Kim S ，Reeves G ，Yeom SI ，Lee JS ，Park M ，Kim S ，Choi IY ，Choi D ，Shin C ... -  《PLoS One》

Identification and characterization of novel microRNAs for fruit development and quality in hot pepper (Capsicum annuum L.).

MicroRNAs (miRNAs) are non-coding small RNAs which play an important regulatory role in various biological processes. Previous studies have reported that miRNAs are involved in fruit development in model plants. However, the miRNAs related to fruit development and quality in hot pepper (Capsicum annuum L.) remains unknown. In this study, small RNA populations from different fruit ripening stages and different varieties were compared using next-generation sequencing technology. Totally, 59 known miRNAs and 310 novel miRNAs were identified from four libraries using miRDeep2 software. For these novel miRNAs, 656 targets were predicted and 402 of them were annotated. GO analysis and KEGG pathways suggested that some of the predicted miRNAs targeted genes involved in starch sucrose metabolism and amino sugar as well as nucleotide sugar metabolism. Quantitative RT-PCR validated the contrasting expression patterns between several miRNAs and their target genes. These results will provide an important foundation for future studies on the regulation of miRNAs involved in fruit development and quality.

Liu Z ，Zhang Y ，Ou L ，Kang L ，Liu Y ，Lv J ，Wei G ，Yang B ，Yang S ，Chen W ，Dai X ，Li X ，Zhou S ，Zhang Z ，Ma Y ，Zou X ... -  《-》

Identification of miRNAs and Their Targets Involved in Flower and Fruit Development across Domesticated and Wild Capsicum Species.

MicroRNAs (miRNAs) are regulators of the post-transcription stage of gene activity documented to play central roles in flower and fruit development in model plant species. However, little is known about their roles and differences in domesticated and wild species. In this study, we used high-throughput sequencing to analyze the miRNA content at three developmental stages (flower, small fruit, and middle fruit) from two cultivated ( and ) and two wild ( and ) pepper species. This analysis revealed 22 known and 27 novel miRNAs differentially expressed across species and tissues. A number of stage- and species-specific miRNAs were identified, and Gene Ontology terms were assigned to 138 genes targeted by the miRNAs. Most Gene Ontology terms were for the categories "genetic information processing", "signaling and cellular processes", "amino acid metabolism", and "carbohydrate metabolism". Enriched KEGG analysis revealed the pathways amino acids, sugar and nucleotide metabolism, starch and sucrose metabolism, and fructose-mannose metabolism among the principal ones regulated by miRNAs during pepper fruit ripening. We predicted miRNA-target gene interactions regulating flowering time and fruit development, including miR156/157 with genes, miR159 with GaMYB proteins, miR160 with ARF genes, miR172 with AP2-like transcription factors, and miR408 with gene across the different species. In addition, novel miRNAs play an important role in regulating interactions potentially controlling plant pathogen defense and fruit quality via fructokinase, alpha-L-arabinofuranosidase, and aromatic and neutral amino acid transporter. Overall, the small RNA-sequencing results from this study represent valuable information that provides a solid foundation for uncovering the miRNA-mediated mechanisms of flower and fruit development between domesticated and wild species.

Lopez-Ortiz C ，Peña-Garcia Y ，Bhandari M ，Abburi VL ，Natarajan P ，Stommel J ，Nimmakayala P ，Reddy UK ... -  《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》

Unravelling the complexity of microRNA-mediated gene regulation in black pepper (Piper nigrum L.) using high-throughput small RNA profiling.

Analysis of high-throughput small RNA deep sequencing data, in combination with black pepper transcriptome sequences revealed microRNA-mediated gene regulation in black pepper ( Piper nigrum L.). Black pepper is an important spice crop and its berries are used worldwide as a natural food additive that contributes unique flavour to foods. In the present study to characterize microRNAs from black pepper, we generated a small RNA library from black pepper leaf and sequenced it by Illumina high-throughput sequencing technology. MicroRNAs belonging to a total of 303 conserved miRNA families were identified from the sRNAome data. Subsequent analysis from recently sequenced black pepper transcriptome confirmed precursor sequences of 50 conserved miRNAs and four potential novel miRNA candidates. Stem-loop qRT-PCR experiments demonstrated differential expression of eight conserved miRNAs in black pepper. Computational analysis of targets of the miRNAs showed 223 potential black pepper unigene targets that encode diverse transcription factors and enzymes involved in plant development, disease resistance, metabolic and signalling pathways. RLM-RACE experiments further mapped miRNA-mediated cleavage at five of the mRNA targets. In addition, miRNA isoforms corresponding to 18 miRNA families were also identified from black pepper. This study presents the first large-scale identification of microRNAs from black pepper and provides the foundation for the future studies of miRNA-mediated gene regulation of stress responses and diverse metabolic processes in black pepper.

Asha S ，Sreekumar S ，Soniya EV 《-》

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.

Taller D ，Bálint J ，Gyula P ，Nagy T ，Barta E ，Baksa I ，Szittya G ，Taller J ，Havelda Z ... -  《PLoS One》