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Possible involvement of locus-specific methylation on expression regulation of leafy homologous gene (CiLFY) during precocious trifoliate orange phase change process.

Zhang JZ, Mei L, Liu R, Khan MR, Hu CG - PLoS ONE (2014)

Bottom Line: Our results showed that flowering locus T (CiFT) relative expression level was increased with the increasing concentrations of 5-AzaC.However, leafy (CiLFY), APETELA1 (CiAP1), terminal flower1 (CiTFL1), and flowering locus C (CiFLC) showed highest relative expression levels at 250 µΜ treatment, while decreased sharply at higher concentrations.These results provided insights into the molecular regulation of CiLFY gene expression, which would be helpful for studying citrus flowering.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Horticultural Plant Biology (Ministry of Education), College of Horticulture and Forestry Science, Huazhong Agricultural University, Wuhan, Hubei Province, China.

ABSTRACT
DNA methylation plays an essential role in regulating plant development. Here, we described an early flowering trifoliate orange (precocious trifoliate orange, Poncirus trifoliata L. Raf) was treated with 5-azacytidine and displayed a number of phenotypic and developmental abnormalities. These observations suggested that DNA methylation might play an important role in regulating many developmental pathways including early flowering trait, and then the expression level of five key or integrated citrus flowering genes were analyzed. Our results showed that flowering locus T (CiFT) relative expression level was increased with the increasing concentrations of 5-AzaC. However, leafy (CiLFY), APETELA1 (CiAP1), terminal flower1 (CiTFL1), and flowering locus C (CiFLC) showed highest relative expression levels at 250 µΜ treatment, while decreased sharply at higher concentrations. In order to further confirm DNA methylation affects the expression of these genes, their full-length sequences were isolated by genome-walker method, and then was analyzed by using bioinformatics tools. However, only one locus-specific methylation site was observed in CiLFY sequence. Therefore, DNA methylation level of the CiLFY was investigated both at juvenile and adult stages of precocious trifoliate orange by bisulfate sequencing PCR; it has been shown that the level of DNA methylation was altered during phase change. In addition, spatial and temporal expression patterns of CiLFY promoter and a series of 5' deletions were investigated by driving the expression of a β-glucuronidase reporter gene in Arabidopsis. Exogenous GA3 treatment on transgenic Arabidopsis revealed that GA3 might be involved in the developmental regulation of CiLFY during flowering process of precocious trifoliate orange. These results provided insights into the molecular regulation of CiLFY gene expression, which would be helpful for studying citrus flowering.

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Statistical analysis of the cytosine methylation status in CpG island and 5′-UTR of CiLFY gene at juvenile and adult phase of precocious trifoliate orange.
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pone-0088558-g004: Statistical analysis of the cytosine methylation status in CpG island and 5′-UTR of CiLFY gene at juvenile and adult phase of precocious trifoliate orange.

Mentions: Locus specific methylation analysis of the CiLFY CpG island was performed to uncover the relationship between the phase change of the precocious trifoliate orange and the methylation status (Figure 4). Observations revealed that the overall DNA methylation status of CiLFY was 25.7% and 18.1% at juvenile and adult stages, respectively. The proportion of three different methylation cytosine for juvenile stage was CG%: 52.3%; CHH%: 14.8%; CHG%: 9.1% and for adult stage was CG%:46.2%; CHH%: 4%; CHG%: 4%, respectively (Figure S1). These results indicated that the prominent methylation decrease during the transition from juvenile stage to adult stage in precocious trifoliate orange. DNA methylation occurs mainly at CG sites in mammals, but these often have been observed at CNG and CHH sites in plants [6], [10]. In this study, CHH and CNG were relative abundant in CiLFY 5′ UTR. Therefore, DNA methylation level of the CiLFY 5′ UTR was also analyzed. Interestingly, bisulfite genomic sequencing results indicated that the overall DNA methylation status of CiLFY 5′-UTR was 6.3% and 6.0% at juvenile and adult stages, respectively (Figure 4). The proportion of three different methylation cytosine was 18.5 and 17.8% (CG); 0.5 and 0% (CHH); and 0 and 0.2% (CHG) at juvenile and adult stages, respectively (Figure S1). Therefore, the demethylation of CiLFY might be the reason for the reflection of gene expression during flower initiation. The results also suggested that there may be an association of CiLFY demethylation to the phage transition in precocious trifoliate orange.


Possible involvement of locus-specific methylation on expression regulation of leafy homologous gene (CiLFY) during precocious trifoliate orange phase change process.

Zhang JZ, Mei L, Liu R, Khan MR, Hu CG - PLoS ONE (2014)

Statistical analysis of the cytosine methylation status in CpG island and 5′-UTR of CiLFY gene at juvenile and adult phase of precocious trifoliate orange.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3921215&req=5

pone-0088558-g004: Statistical analysis of the cytosine methylation status in CpG island and 5′-UTR of CiLFY gene at juvenile and adult phase of precocious trifoliate orange.
Mentions: Locus specific methylation analysis of the CiLFY CpG island was performed to uncover the relationship between the phase change of the precocious trifoliate orange and the methylation status (Figure 4). Observations revealed that the overall DNA methylation status of CiLFY was 25.7% and 18.1% at juvenile and adult stages, respectively. The proportion of three different methylation cytosine for juvenile stage was CG%: 52.3%; CHH%: 14.8%; CHG%: 9.1% and for adult stage was CG%:46.2%; CHH%: 4%; CHG%: 4%, respectively (Figure S1). These results indicated that the prominent methylation decrease during the transition from juvenile stage to adult stage in precocious trifoliate orange. DNA methylation occurs mainly at CG sites in mammals, but these often have been observed at CNG and CHH sites in plants [6], [10]. In this study, CHH and CNG were relative abundant in CiLFY 5′ UTR. Therefore, DNA methylation level of the CiLFY 5′ UTR was also analyzed. Interestingly, bisulfite genomic sequencing results indicated that the overall DNA methylation status of CiLFY 5′-UTR was 6.3% and 6.0% at juvenile and adult stages, respectively (Figure 4). The proportion of three different methylation cytosine was 18.5 and 17.8% (CG); 0.5 and 0% (CHH); and 0 and 0.2% (CHG) at juvenile and adult stages, respectively (Figure S1). Therefore, the demethylation of CiLFY might be the reason for the reflection of gene expression during flower initiation. The results also suggested that there may be an association of CiLFY demethylation to the phage transition in precocious trifoliate orange.

Bottom Line: Our results showed that flowering locus T (CiFT) relative expression level was increased with the increasing concentrations of 5-AzaC.However, leafy (CiLFY), APETELA1 (CiAP1), terminal flower1 (CiTFL1), and flowering locus C (CiFLC) showed highest relative expression levels at 250 µΜ treatment, while decreased sharply at higher concentrations.These results provided insights into the molecular regulation of CiLFY gene expression, which would be helpful for studying citrus flowering.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Horticultural Plant Biology (Ministry of Education), College of Horticulture and Forestry Science, Huazhong Agricultural University, Wuhan, Hubei Province, China.

ABSTRACT
DNA methylation plays an essential role in regulating plant development. Here, we described an early flowering trifoliate orange (precocious trifoliate orange, Poncirus trifoliata L. Raf) was treated with 5-azacytidine and displayed a number of phenotypic and developmental abnormalities. These observations suggested that DNA methylation might play an important role in regulating many developmental pathways including early flowering trait, and then the expression level of five key or integrated citrus flowering genes were analyzed. Our results showed that flowering locus T (CiFT) relative expression level was increased with the increasing concentrations of 5-AzaC. However, leafy (CiLFY), APETELA1 (CiAP1), terminal flower1 (CiTFL1), and flowering locus C (CiFLC) showed highest relative expression levels at 250 µΜ treatment, while decreased sharply at higher concentrations. In order to further confirm DNA methylation affects the expression of these genes, their full-length sequences were isolated by genome-walker method, and then was analyzed by using bioinformatics tools. However, only one locus-specific methylation site was observed in CiLFY sequence. Therefore, DNA methylation level of the CiLFY was investigated both at juvenile and adult stages of precocious trifoliate orange by bisulfate sequencing PCR; it has been shown that the level of DNA methylation was altered during phase change. In addition, spatial and temporal expression patterns of CiLFY promoter and a series of 5' deletions were investigated by driving the expression of a β-glucuronidase reporter gene in Arabidopsis. Exogenous GA3 treatment on transgenic Arabidopsis revealed that GA3 might be involved in the developmental regulation of CiLFY during flowering process of precocious trifoliate orange. These results provided insights into the molecular regulation of CiLFY gene expression, which would be helpful for studying citrus flowering.

Show MeSH
Related in: MedlinePlus