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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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Histochemical localization of GUS activity in transgenic Arabidopsis.a–d: seedlings at 7, 10, 14 and 20 days, respectively; e: lateral flower bud just emerged; f: lateral flower bud; g: apex flower bud; h: young inflorescence; i: fully-opened flower; j: young leaves; k old leaves; l: roots from adult plants; m: fruit. bar: 1 mM.
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pone-0088558-g005: Histochemical localization of GUS activity in transgenic Arabidopsis.a–d: seedlings at 7, 10, 14 and 20 days, respectively; e: lateral flower bud just emerged; f: lateral flower bud; g: apex flower bud; h: young inflorescence; i: fully-opened flower; j: young leaves; k old leaves; l: roots from adult plants; m: fruit. bar: 1 mM.

Mentions: To examine the tissue specificity of the CiLFY promoter, a GUS expression construct driven by full-length CiLFY promoter (1,641 bp) was introduced into Arabidopsis (Figure 5). Transgenic plants were confirmed for the presence of appropriate expression cassette by PCR. Overall, histochemical analysis have indicated that the GUS enzyme activity was detected in adult roots, young leaves, floral petals and sepals, but was not in the pistil, stamens, or seeds (Figure 5). At juvenile stage, GUS expression was first detected in 7-day-old seedlings, there was stronger GUS staining in the first two true leaves, while there was relatively weaker staining in the hypocotyls, but no staining was observed in cotyledon and radicles. Similar GUS activities were maintained in 14-day-old aerial parts (Figure 5). Histochemical assay at adult stage indicated that GUS staining was found in all floral buds (including apex and lateral). Detailed flower study has revealed that the sepals, peripheral petal, and stigmatic papillae exhibited very intense GUS expression (Figure 5), while anther locules and stamen filaments did not exhibit any GUS expression (Figure 5). The other aerial plant parts like stem, bracts and mature leaves did not exhibit any GUS expression. In fruit, GUS expression was only confined to fruit abscission zone. The GUS expression pattern was also corroborated with the pattern observed by real-time PCR (Figure 3B). All of these results indicated that the CiLFY promoter modulated precise transcriptional regulation of specific and developmental expression in transgenic Arabidopsis.


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)

Histochemical localization of GUS activity in transgenic Arabidopsis.a–d: seedlings at 7, 10, 14 and 20 days, respectively; e: lateral flower bud just emerged; f: lateral flower bud; g: apex flower bud; h: young inflorescence; i: fully-opened flower; j: young leaves; k old leaves; l: roots from adult plants; m: fruit. bar: 1 mM.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0088558-g005: Histochemical localization of GUS activity in transgenic Arabidopsis.a–d: seedlings at 7, 10, 14 and 20 days, respectively; e: lateral flower bud just emerged; f: lateral flower bud; g: apex flower bud; h: young inflorescence; i: fully-opened flower; j: young leaves; k old leaves; l: roots from adult plants; m: fruit. bar: 1 mM.
Mentions: To examine the tissue specificity of the CiLFY promoter, a GUS expression construct driven by full-length CiLFY promoter (1,641 bp) was introduced into Arabidopsis (Figure 5). Transgenic plants were confirmed for the presence of appropriate expression cassette by PCR. Overall, histochemical analysis have indicated that the GUS enzyme activity was detected in adult roots, young leaves, floral petals and sepals, but was not in the pistil, stamens, or seeds (Figure 5). At juvenile stage, GUS expression was first detected in 7-day-old seedlings, there was stronger GUS staining in the first two true leaves, while there was relatively weaker staining in the hypocotyls, but no staining was observed in cotyledon and radicles. Similar GUS activities were maintained in 14-day-old aerial parts (Figure 5). Histochemical assay at adult stage indicated that GUS staining was found in all floral buds (including apex and lateral). Detailed flower study has revealed that the sepals, peripheral petal, and stigmatic papillae exhibited very intense GUS expression (Figure 5), while anther locules and stamen filaments did not exhibit any GUS expression (Figure 5). The other aerial plant parts like stem, bracts and mature leaves did not exhibit any GUS expression. In fruit, GUS expression was only confined to fruit abscission zone. The GUS expression pattern was also corroborated with the pattern observed by real-time PCR (Figure 3B). All of these results indicated that the CiLFY promoter modulated precise transcriptional regulation of specific and developmental expression in transgenic Arabidopsis.

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