Table 1.
PCR primers used in this study.
Fig 1.
Phenotypes of MIGS-chs transgenic flowers.
(A) V26 (wild-type, left) and transgenic (right, L1) flowers. (B and C) qRT-PCR detection of mRNA levels of CHS-A and CHS-J genes in V26 and in transgenic line 1 (L1). (D) ‘Carpet Purple’ (wild-type, left) and transgenic (right, Lc3) flowers. (E and F) qRT-PCR detection of mRNA levels of CHS-A and CHS-J genes in ‘Carpet Purple’ (CP) and in transgenic line c3 (Lc3).
Fig 2.
Phenotypes of MIGS-pds transgenic callus and shoots.
(A) MD (left) and transgenic (right) petunia callus. (B) MD (right) and transgenic (left) shoots. (C) qRT-PCR detection of mRNA levels of PDS genes in MD petunia transgenic lines (T1 and T12).
Fig 3.
Mapping of miR173- and siRNA-mediated cleavage sites using modified 5′ RLM-RACE.
(A) miR173-mediated cleavage of miR173ts_CHS transcripts. The cleavage position and the number of sequenced clones corresponding to the site are indicated by a vertical arrow. (B) Agarose gels showing the products of the mapping of cleavage sites of CHAS-A and CHS-J mRNAs. (C) Cleavage of CHS-A mRNA. (D) Cleavage of CHS-J mRNA. The cleavage sites and their corresponding positions on genomic sequences (x14591 and x14597) are indicated by vertical arrows. Horizontal bars represent transcripts. Horizontal arrows indicate the position of gene specific primers used in RACE PCR. TL: tobacco etch virus leader sequence.
Fig 4.
Processing of miR173 precursors.
Small RNA sequences from (A) MIGS-chs transgenic petunia petals and (B) Arabidopsis were incorporated into the predicted stem-loop of the Arabidopsis miR173 precursor. Arabidopsis small RNAs were retrieved from the miRBase database (v21). MiR173 is denoted by pink and miR173* by cyan. The read count is indicated beside each small RNA species and only small RNAs cloned more than five times are indicated. Small RNA read counts from Arabidopsis totaled 1,485 and from MIGS-chs transgenic petunia the total was 3,190.
Fig 5.
Position and abundance of small RNAs matching the miR173ts_CHS transcripts, endogenous CHS-A and CHS-J.
(A) 21-nt and (B) 22-nt siRNAs mapped onto the miR173ts_CHS transcripts. (C) 21-nt siRNAs mapped onto the CHS-A. (D) 21-nt siRNAs specific for CHS-J. Vertical bars above and below the horizontal bar (x-axis) denote small RNAs originate from sense and antisense dsRNA strands, respectively.
Fig 6.
Phase distribution of small RNAs.
(A) Phase distribution and abundance of 21-nt small RNAs mapped onto the miR173ts_CHS transcripts. Paired sense and antisense 21-nt RNAs with 2-nt 3′ overhangs are consolidated to one small RNA unit. Processing cycles augment at 21-nt intervals. The first phase position in each processing cycle is in-phase with the miR173 register, and is highlighted in red and indicated by stars if it is populated by at least one small RNA read. (B) Small RNAs mapped onto the first four processing cycles downstream from the miR173 cleavage site. The short horizontal lines indicate 21-nt small RNAs. Their corresponding read counts are shown above each line. Red and blue lines denote those small RNAs in phase with the miR173 register with respect to sense and antisense dsRNA strands, respectively. Only small RNAs cloned more than 5 times are indicated.