Figure 1.
Promoter sequences of PeMADS2∼6 and the putative CArG boxes.
Length of promoter sequences of PeMADS2, PeMADS3, and PeMADS6 were 3,249, 1,293 and 1,514 bp, respectively. The promoter sequence of the PeMADS4 was extended from −422 bp to −3,303 bp (horizontal line box), and the original fragment of −2,121 to −840 bp of PeMADS5 promoter was replaced by a 1,227-bp fragment (diagonal line box). The rhombus and the triangles indicate the putative CArG boxes predicted by consensus CC(A/T)6GG and C(A/T)8G motifs, respectively. “+1” means the transcription start site and ATG was the translation start site. The lengths of the promoters are show from the transcription start site to the upstream sequences.
Table 1.
BAC clones containing various PeMADS genes.
Table 2.
Putative CArG boxes at the PeMADS promoter regions predicted by homemade software and the PLACE database.
Figure 2.
Visual representation of motifs in the 1.3-kb promoter sequences of PeMADS2∼5.
FOOTPRINTER parameters: (A) motif size: 11, allowed mutations: 0, (B) motif size: 10, allowed mutations: 0. (C) Putative CArG boxes in the PeMADS promoter regions predicted with the homemade software (red box) and the PLACE database (blue box).
Figure 3.
GUS histochemical staining for the promoter activities of PeMADS2∼6.
Histochemical assay of GUS expression in floral organs shown in the order of pBI-Pe2p-3249 (A–D), pBI-Pe3p-1293 (E–H), pBI-Pe4p-3303 (I–L), pBI-Pe5p-2062 (M–P), pBI-Pe6p-1514 (Q–T), pBI221 (U–X) and pBI-PL (Y-AB). Constructs were bombarded into four independent floral buds, and results are representative of three independent bombardment experiments. Scale bar = 0.5 mm.
Figure 4.
Functional analysis of serial deletions of PeMADS6 promoter.
(A) Serial deletion constructs of PeMADS6 promoter. (B-Q) Histochemical assay of flower organs bombarded with serial deletions of PeMADS6 promoter shown in the order of pBI-Pe6p-1108 (B–E), pBI-Pe6p-808 (F–I), pBI-Pe6p-508 (J–M) and pBI-Pe6p-208 (N–Q). Constructs were bombarded into four independent floral buds, and results are representative of three independent bombardment experiments. Scale bar = 0.5 mm. (R) Dual luciferase assay of serial deletions of PeMADS6 promoter. The same letters above the bars are not statistically different by T-test analysis (p<0.01). Data are mean ± SD (n = 6). All constructs were analyzed for promoter activities of driving luciferase expression by bombardment into two floral buds, and results are representative of three independent bombardment experiments.
Figure 5.
Functional analysis of serial deletions of PeMADS4 promoter.
(A) Serial deletion constructs of PeMADS4 promoter. (B–Q) Histochemical assay of flower organs bombarded with serial deletions of PeMADS4 promoter shown in the order of pBI-Pe4p-2313 (B–E), pBI-Pe4p-1497 (F–I), pBI-Pe4p-935 (J–M) and pBI-Pe4p-375 (N–Q). Constructs were bombarded into four independent floral buds, and results are representative of three independent bombardment experiments. Scale bar = 0.5 mm. (R) Dual luciferase assay of the serial deletions of PeMADS4 promoter. The same letters above the bars are not statistically different by T-test analysis (p<0.01). Data are mean ± SD (n = 6). All constructs were analyzed for promoter activities of driving luciferase expression by bombardment into two floral buds, and results are representative of three independent bombardment experiments.
Figure 6.
Histochemical assay of the 5th intron of PeMADS4.
(A) Genomic structure of PeMADS4. Gray, black, and white boxes indicate the promoter, exon, and intron regions of PeMADS4 gene, respectively. Numbers above the black boxes are the number and length (bp) of exons, respectively. Numbers beneath the white boxes are the number and length (bp) of introns, respectively. Two CC(A/T)6GG sequences (rhombus) and 11 C(A/T)8G sequences (triangles) are located in the 5th intron. Three serial deletions of the 5th intron were designed for 2-, 3.5- and 8-kb sequences, respectively, and inserted into the upstream region of the Pe4pF1 promoter sequence in the pBI-Pe4p-375 construct. (B-Q) Histochemical assay of the serial deletions of the 5th intron of PeMADS4 were in the order of pBI-Pe4p-375 (B–E), pBI-Pe4p-375-8- (F–I), pBI-Pe4p-375-3.5- (J–M) and pBI-Pe4p-375-2-kb 5th intron constructs (N–Q). Constructs were bombarded into four independent floral buds, and results are representative of three independent bombardment experiments. Scale bar = 0.5 mm.
Figure 7.
Methylation status in the promoter and 5th intron regions of PeMADS4.
(A) Locations of the probes used for Southern blot analysis and methylation status in the promoter (B) and 5th intron (C) regions of PeMADS4. Gray, black, and white boxes indicate the promoter, exon, and intron regions of PeMADS4 gene, respectively. The rhombus and black triangles are the predicted CArG boxes. The white triangles point to the HpaII/MspI sites. Probes used in this study are shown in gray located in the 5′ UTR (Probe 1) or in the 5th intron (Probe 2). (B) Southern blot analysis was performed with genomic DNA extracted from petal and lip of P. equestris, digested with methylation-sensitive enzymes, HpaII (H) and MspI (M), and hybridized with probe 1. (C) Southern blot analysis was performed with the genomic DNA extracted from sepals, petals, lips and columns of P. equestris, double-digested with DraI and HpaII, and hybridized with probe 2. Probe 2 was a 2,136-bp fragment between three DraI restriction enzyme cleavage sites and contained two HpaII site. (D) Bisultife sequencing for the methylation status within the promoter region of PeMADS4.
Figure 8.
Histone modification on the ATG and 5th intron regions of PeMADS4.
(A) Locations of the primers used in ChIP assay. Gray, black, and white boxes indicate the promoter, exon, and intron regions of PeMADS4 gene, respectively. The rhombus and black triangles are the predicted CArG boxes. ChIP assay of histone modification of dimethyl-H3K9 (H3K9me2), trimethyl-H3K4 (H3K4me3), acetyl-H3K9 and H3K14 (H3Ac) was analyzed on the ATG (B) and 5th intron regions (C) of PeMADS4 in the petal and lip of P. equestris. The amount of DNA after ChIP was quantified and normalized to an internal control ACTIN2 for H3K4me3 and H3K9K14ac or Ta3 for H3K9me2. Data are mean ± SD calculated from three technological and two biological replicates. X = fold.