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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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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.
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pone-0106033-g004: 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.

Mentions: The PI-like PeMADS6 was expressed ubiquitously in all floral organs. To assess the minimal promoter region of PeMADS6, four deletion clones of the PeMADS6 promoter sequence were resulted, including 1,108-bp, 808-bp, 508-bp, and 208-bp fragments containing 3, 2, 0, and 0 CArG boxes, respectively. Similar to the full length PeMADS6 promoter construct, pBI-Pe6p-1514 (Fig. 3Q-T), all four deletion promoter sequences of PeMADS6 could drive GUS expression in all the floral organs examined (Fig. 4B–Q), although the expression was slightly decreased in pBI-Pe6p-508 and pBI-Pe6p-208 constructs (Fig. 4J–Q). Moreover, quantitative dual luciferase assay was performed to further examine the differential promoter activities of these serial deletion constructs. The pJD-Pe6p-208 construct was sufficient to drive luciferase expression in all floral organs (Fig. 4R). Extension of the upstream sequence from the pJD-Pe6p-508 to pJD-Pe6p-1514 constructs conferred similar GUS expression in all floral organs (Fig. 4R). Therefore, the 208-bp promoter sequence was a minimal promoter for PeMADS6 expression in all four floral organs.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4263434&req=5

pone-0106033-g004: 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.
Mentions: The PI-like PeMADS6 was expressed ubiquitously in all floral organs. To assess the minimal promoter region of PeMADS6, four deletion clones of the PeMADS6 promoter sequence were resulted, including 1,108-bp, 808-bp, 508-bp, and 208-bp fragments containing 3, 2, 0, and 0 CArG boxes, respectively. Similar to the full length PeMADS6 promoter construct, pBI-Pe6p-1514 (Fig. 3Q-T), all four deletion promoter sequences of PeMADS6 could drive GUS expression in all the floral organs examined (Fig. 4B–Q), although the expression was slightly decreased in pBI-Pe6p-508 and pBI-Pe6p-208 constructs (Fig. 4J–Q). Moreover, quantitative dual luciferase assay was performed to further examine the differential promoter activities of these serial deletion constructs. The pJD-Pe6p-208 construct was sufficient to drive luciferase expression in all floral organs (Fig. 4R). Extension of the upstream sequence from the pJD-Pe6p-508 to pJD-Pe6p-1514 constructs conferred similar GUS expression in all floral organs (Fig. 4R). Therefore, the 208-bp promoter sequence was a minimal promoter for PeMADS6 expression in all four floral organs.

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
Related in: MedlinePlus