Zhang Jin-Zhi, Mei Li, Liu Rong, Khan Muhammad Rehman Gul, Hu Chun-Gen
Key Laboratory of Horticultural Plant Biology (Ministry of Education), College of Horticulture and Forestry Science, Huazhong Agricultural University, Wuhan, Hubei Province, China.
PLoS One. 2014 Feb 11;9(2):e88558. doi: 10.1371/journal.pone.0088558. eCollection 2014.
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.
DNA甲基化在调控植物发育过程中起着至关重要的作用。在此，我们描述了一种早花枳（早熟枳，Poncirus trifoliata L. Raf），用5-氮杂胞苷处理后表现出许多表型和发育异常。这些观察结果表明，DNA甲基化可能在调控包括早花性状在内的许多发育途径中发挥重要作用，随后分析了五个关键或整合的柑橘开花基因的表达水平。我们的结果表明，随着5-氮杂胞苷浓度的增加，开花位点T（CiFT）的相对表达水平升高。然而，叶状（CiLFY）、APETELA1（CiAP1）、顶花1（CiTFL1）和开花位点C（CiFLC）在250 μΜ处理时显示出最高的相对表达水平，而在更高浓度下急剧下降。为了进一步证实DNA甲基化影响这些基因的表达，通过基因组步移法分离了它们的全长序列，然后使用生物信息学工具进行分析。然而，在CiLFY序列中仅观察到一个位点特异性甲基化位点。因此，通过亚硫酸氢盐测序PCR研究了早熟枳幼年和成年阶段CiLFY的DNA甲基化水平；结果表明，DNA甲基化水平在阶段转变过程中发生了改变。此外，通过在拟南芥中驱动β-葡萄糖醛酸酶报告基因的表达，研究了CiLFY启动子的时空表达模式以及一系列5'缺失。对转基因拟南芥进行外源GA3处理表明，GA3可能参与早熟枳开花过程中CiLFY的发育调控。这些结果为CiLFY基因表达的分子调控提供了见解，这将有助于研究柑橘开花。
