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

Abstract

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 Capsicum 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 (C. baccatum and C. annuum) and two wild (C. chacoense and C. eximium) 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 SPL genes, miR159 with GaMYB proteins, miR160 with ARF genes, miR172 with AP2-like transcription factors, and miR408 with CLAVATA1 gene across the different Capsicum 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 Capsicum species.

Keywords: Capsicum species; flower; fruit development; miRNA.

Figure 1

Morphological features of pepper fruit development stages of four Capsicum species. Harvested fruit at different developmental stages from days after pollination of (a) C. baccatum, (b) C. annuum, (c) C. chacoense, and (d) C. eximium. Red arrows indicate small fruit (6-dpa) and middle fruit (25-dpa) stages that were collected for small RNA sequencing.

Figure 2

Length distribution of small RNAs in (a) C. baccatum, (b) C. annuum, (c) C. chacoense, and (d) C. eximium at flowering, small, and medium fruit stages.

Figure 3

Predicted secondary hairpin structures of six novel miRNAs identified in Capsicum species. Base pair probability is represented by color: red, high-probability, and purple, low-probability.

Figure 4

Differential expression analysis of known miRNAs. (a) Heat map of known miRNA expression profiles in C. baccatum (Cb), C. annuum (Ca), C. chacoense (Cch), and C. eximium (Ce) at different stages analyzed. The expression levels are represented by the color: red, high-expressed; and blue, low-expressed. Venn diagram analysis of shared known miRNAs at (b) flowering, (c) small, and (d) medium fruit stages across Capsicum species.

Figure 5

Differential expression analysis of novel miRNAs. (a) Heat map of novel miRNA expression profiles in C. baccatum (Cb), C. annuum (Ca), C. chacoense (Cch), and C. eximium (Ce) at different stages analyzed. The expression levels are represented by the color: red, high-expressed; and blue, low-expressed. Venn diagram analysis of shared novel miRNAs at (b) flowering, (c) small, and (d) medium fruit stages across Capsicum species.

Figure 6

Expression patterns of known and novel miRNA target genes in leaf, stem, and placenta (PL) tissue at 6, 16, and 25 days post-anthesis of C. annuum var CM344. The expression levels are represented by color: red, upregulated; and blue, downregulated.

Figure 7

Validation of six known and six novel miRNAs by stem-loop RT-qPCR in Capsicum species at flowering, small fruit, and middle fruit stages. The small nuclear RNA (snRNA) U6 was a housekeeping gene. Data are mean ± SD from three biological replicates.

Figure 8

Expression of target genes of known and novel miRNAs in Capsicum species at flowering, small fruit, and middle fruit stages. B-tub gene was a housekeeping gene. Data are mean ± SD from three biological replicates.