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Histone acetylation accompanied with promoter sequences displaying differential expression profiles of B-class MADS-box genes for phalaenopsis floral morphogenesis.

Hsu CC, Wu PS, Chen TC, Yu CW, Tsai WC, Wu K, Wu WL, Chen WH, Chen HH - PLoS ONE (2014)

Bottom Line: The amplified promoter sequences of PeMADS2∼6 could drive beta-glucuronidase (GUS) gene expression in all floral organs, similar to their expression at the floral primordia stage.The promoter sequence of PeMADS4, exclusively expressed in lip and column, showed a 1.6∼3-fold higher expression in lip/column than in sepal/petal.Furthermore, we noted a 4.9-fold increase in histone acetylation (H3K9K14ac) in the translation start region of PeMADS4 in lip as compared in petal.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, National Cheng Kung University, Tainan, Taiwan.

ABSTRACT
Five B-class MADS-box genes, including four APETALA3 (AP3)-like PeMADS2∼5 and one PISTILLATA (PI)-like PeMADS6, specify the spectacular flower morphology in orchids. The PI-like PeMADS6 ubiquitously expresses in all floral organs. The four AP3-like genes, resulted from two duplication events, express ubiquitously at floral primordia and early floral organ stages, but show distinct expression profiles at late floral organ primordia and floral bud stages. Here, we isolated the upstream sequences of PeMADS2∼6 and studied the regulatory mechanism for their distinct gene expression. Phylogenetic footprinting analysis of the 1.3-kb upstream sequences of AP3-like PeMADS2∼5 showed that their promoter regions have sufficiently diverged and contributed to their subfunctionalization. The amplified promoter sequences of PeMADS2∼6 could drive beta-glucuronidase (GUS) gene expression in all floral organs, similar to their expression at the floral primordia stage. The promoter sequence of PeMADS4, exclusively expressed in lip and column, showed a 1.6∼3-fold higher expression in lip/column than in sepal/petal. Furthermore, we noted a 4.9-fold increase in histone acetylation (H3K9K14ac) in the translation start region of PeMADS4 in lip as compared in petal. All these results suggest that the regulation via the upstream sequences and increased H3K9K14ac level may act synergistically to display distinct expression profiles of the AP3-like genes at late floral organ primordia stage for Phalaenopsis floral morphogenesis.

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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.
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pone-0106033-g006: 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.

Mentions: The longest introns of AG and FLC in Arabidopsis play a regulatory role for their gene expression [12], [13]. It was intriguing to know whether the introns of PeMADS4 may regulate its distinct expression at the late floral organ primordia stage of Phalaenopsis orchids. To test this, we first sequenced the genomic sequence of BAC clones containing PeMADS4, NCKU-PE-btBAC-1105 H24. Then, the genomic sequence was compared to its cDNA sequence, and seven exons and six introns were identified for PeMADS4 with a long 5th intron of 9,483 bp (Fig. 6A). The 5th intron contains two conserved CC(A/T)6GG motifs (Fig. 6A, rhombus) and 11 C(A/T)8G motifs (Fig. 6A, triangles) (Fig. 6A).


Histone acetylation accompanied with promoter sequences displaying differential expression profiles of B-class MADS-box genes for phalaenopsis floral morphogenesis.

Hsu CC, Wu PS, Chen TC, Yu CW, Tsai WC, Wu K, Wu WL, Chen WH, Chen HH - PLoS ONE (2014)

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.
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Related In: Results  -  Collection

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pone-0106033-g006: 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.
Mentions: The longest introns of AG and FLC in Arabidopsis play a regulatory role for their gene expression [12], [13]. It was intriguing to know whether the introns of PeMADS4 may regulate its distinct expression at the late floral organ primordia stage of Phalaenopsis orchids. To test this, we first sequenced the genomic sequence of BAC clones containing PeMADS4, NCKU-PE-btBAC-1105 H24. Then, the genomic sequence was compared to its cDNA sequence, and seven exons and six introns were identified for PeMADS4 with a long 5th intron of 9,483 bp (Fig. 6A). The 5th intron contains two conserved CC(A/T)6GG motifs (Fig. 6A, rhombus) and 11 C(A/T)8G motifs (Fig. 6A, triangles) (Fig. 6A).

Bottom Line: The amplified promoter sequences of PeMADS2∼6 could drive beta-glucuronidase (GUS) gene expression in all floral organs, similar to their expression at the floral primordia stage.The promoter sequence of PeMADS4, exclusively expressed in lip and column, showed a 1.6∼3-fold higher expression in lip/column than in sepal/petal.Furthermore, we noted a 4.9-fold increase in histone acetylation (H3K9K14ac) in the translation start region of PeMADS4 in lip as compared in petal.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, National Cheng Kung University, Tainan, Taiwan.

ABSTRACT
Five B-class MADS-box genes, including four APETALA3 (AP3)-like PeMADS2∼5 and one PISTILLATA (PI)-like PeMADS6, specify the spectacular flower morphology in orchids. The PI-like PeMADS6 ubiquitously expresses in all floral organs. The four AP3-like genes, resulted from two duplication events, express ubiquitously at floral primordia and early floral organ stages, but show distinct expression profiles at late floral organ primordia and floral bud stages. Here, we isolated the upstream sequences of PeMADS2∼6 and studied the regulatory mechanism for their distinct gene expression. Phylogenetic footprinting analysis of the 1.3-kb upstream sequences of AP3-like PeMADS2∼5 showed that their promoter regions have sufficiently diverged and contributed to their subfunctionalization. The amplified promoter sequences of PeMADS2∼6 could drive beta-glucuronidase (GUS) gene expression in all floral organs, similar to their expression at the floral primordia stage. The promoter sequence of PeMADS4, exclusively expressed in lip and column, showed a 1.6∼3-fold higher expression in lip/column than in sepal/petal. Furthermore, we noted a 4.9-fold increase in histone acetylation (H3K9K14ac) in the translation start region of PeMADS4 in lip as compared in petal. All these results suggest that the regulation via the upstream sequences and increased H3K9K14ac level may act synergistically to display distinct expression profiles of the AP3-like genes at late floral organ primordia stage for Phalaenopsis floral morphogenesis.

Show MeSH