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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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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.
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pone-0106033-g007: 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.

Mentions: To examine whether the specific expression of PeMADS4 in lip and column was caused by DNA methylation in the regulatory sequences in planta, Southern blot hybridization was performed to analyze the DNA methylation status in the promoter, translation start site, and intron regions of PeMADS4. DNA samples isolated from petal and lip of P. equestris were digested with the methylation-sensitive endonucleases HpaII or MspI (H or M) (Fig. 7B) and DraI/HpaII (Fig. 7C). Probe 1 containing a 582-bp fragment including a 375-bp promoter sequence and a 207-bp 5′-UTR region of PeMADS4 (Fig. 7A, B) was used for DNA samples from petal or lip and similar methylation status were obtained with digestion of HpaII (Fig. 7B, 4.2- and 8-kb fragments) and MspI (Fig. 7B, 4.2- and 5.6-kb fragments). Moreover, hybridization with probe 2, containing a 2,136-bp fragment of the 5th intron of PeMADS4, gave the same results for all DraI/HpaII-digested DNA samples from sepal, petal, lip, and column (Fig. 7A, C, 1- and 1.2-kb fragments), so the HpaII site within probe 2 region was methylated in all four floral organs. Thus, the DNA methylation status was the same in the promoter region, translation start site, and the 5th intron regions of the PeMADS4 gene for both petal and lip of Phalaenopsis flowers.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0106033-g007: 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.
Mentions: To examine whether the specific expression of PeMADS4 in lip and column was caused by DNA methylation in the regulatory sequences in planta, Southern blot hybridization was performed to analyze the DNA methylation status in the promoter, translation start site, and intron regions of PeMADS4. DNA samples isolated from petal and lip of P. equestris were digested with the methylation-sensitive endonucleases HpaII or MspI (H or M) (Fig. 7B) and DraI/HpaII (Fig. 7C). Probe 1 containing a 582-bp fragment including a 375-bp promoter sequence and a 207-bp 5′-UTR region of PeMADS4 (Fig. 7A, B) was used for DNA samples from petal or lip and similar methylation status were obtained with digestion of HpaII (Fig. 7B, 4.2- and 8-kb fragments) and MspI (Fig. 7B, 4.2- and 5.6-kb fragments). Moreover, hybridization with probe 2, containing a 2,136-bp fragment of the 5th intron of PeMADS4, gave the same results for all DraI/HpaII-digested DNA samples from sepal, petal, lip, and column (Fig. 7A, C, 1- and 1.2-kb fragments), so the HpaII site within probe 2 region was methylated in all four floral organs. Thus, the DNA methylation status was the same in the promoter region, translation start site, and the 5th intron regions of the PeMADS4 gene for both petal and lip of Phalaenopsis flowers.

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