Fig 1.
Developmental stages of the sweet pepper cv. Fehérözön investigated in this study.
(A) The two selected fruit developmental stages at 28 and 40 days after anthesis (DAA). Grid = 1 cm. (B) Vertical and horizontal sections of expanding fruit showing the composing tissues as indicated. The seeds, the placenta, and the flesh were isolated and used for RNA extractions separately.
Table 1.
Summary of the read numbers along the filtering steps in the small RNA libraries.
Fig 2.
Size distribution of small RNAs in the sequencing libraries.
The diagram shows the small RNA size composition of each library. Biological replicates are displayed next to each other. Numbers at the top indicate the length of sRNAs.
Fig 3.
(A) Read alignment diagram for the selected miRNA precursors. Mature strand is highlighted with green and star strand with red. (B) Predicted secondary structure of pre-miRNAs (Mfold). Mature miRNA and miRNA* sequences are highlighted.
Fig 4.
Small RNA Northern blot analyses of selected miRNAs.
Expression of the conservative (green) and the newly predicted (red) miRNAs. The total RNA samples from the seed, the placenta and the flesh at 28 and 40 days after anthesis (DAA) were run on agarose gels, transferred to nylon membranes, and hybridized with probes detecting miRNAs as indicated. We show the NGS read counts (read/million) in the upper panels, the results of small RNA northern blots in the middle panels, and the rRNA levels as loading controls in the lower panels. We used the same membrane for the hybridization of miR171, miR396, and miR159, can-mir-f13 specific probes.
Fig 5.
Heat map showing the expression levels of the differentially expressed miRNAs.
The differential expression analysis between the seed (S), the placenta (P), and the flesh (F) samples at (A) 28 DAA and (B) 40 DAA was conducted using DESeq2. The results were filtered to keep only those miRNAs whose mean normalized expression level was at least 10, and the expression change was at least six-fold. We colored the conservative (black), known (blue) and novel (red) miRNAs. We found 21 differentially expressed miRNAs between different stages (28, 40) in the flesh (C), 28 miRNAs in the seed (D), and none in the placenta. Arrows represent up- (↑) and down-regulation (↓) compared to the two other tissues (for example S↑ means, the expression of the marked miRNA are upregulated in seed compared to both, placenta and flesh. We also listed those changes, which occurred only between two tissues. For example, we marked a miRNA up-regulation between the placenta and the seed like this: P→S↑.
Fig 6.
Accumulation of AGO1 protein and miR168 in pepper fruit tissues.
RNA and protein samples of seed, placenta, and flesh, representing developmental stages at 28 and 40 days after anthesis (DAA), were used for small RNA northern blot analyses and Western blotting. Upper panel is the schematic representation of miR168 NGS read counts (read/million). Middle panels show the results miR168 northern blots. EtBr stained rRNAs serve as loading control. Bottom panels show Western blot analyses of total protein extracts for AGO1 accumulation. Equal loading was verified by Ponceau staining of the membrane after western blotting.
Fig 7.
Expression pattern and sequence length composition of 24-nt-long hetsiRNA-producing clusters.
Heat map showing (A) the log2 transformed expression levels (Read Per Million) and (B) sequence length composition of the 50 most abundant clusters, and (C) Northern blot analyses of the expression of Cluster 297979 and Cluster 461037.