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

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.