Possible involvement of DNA methylation on expression regulation of carrot LEC1 gene in its 5'-upstream region.

DNA methylation plays important roles in various developmental processes in many organisms. In carrots, the treatment of embryogenic cells (ECs) with DNA methylation inhibitors induces hypomethylation and blocks somatic embryogenesis. CARROT-LEAFY COTYLEDON 1 (C-LEC1) is an important transcription factor for embryo development that shows embryo-specific expression in ECs and somatic and zygotic embryos. However, the regulation of embryo-specific transcription factor genes such as C-LEC1 in plants is not well understood. In this study, we used embryogenic carrot cells (Daucus carota L. cv. US-Harumakigosun) to investigate the DNA methylation status of the embryogenesis-related genes C-LEC1, Carrot ABA INSENSITIVE 3 (C-ABI3), and Daucus carota Embryogenic cell protein 31 (DcECP 31) during the transition from embryogenesis to vegetative growth. The C-LEC1 promoter region showed a reduced level of DNA methylation during somatic embryogenesis followed by an increase during the transition from embryonic to vegetative growth. To test whether the increased level of DNA methylation down-regulates C-LEC1 expression, RNA-directed DNA methylation (RdDM) was used to induce the hypermethylation of two segments of the C-LEC1 5'-upstream region: Regions 1 and 2, corresponding to nucleotides -1,904 to -1,272 and -896 to -251, respectively. When the hypermethylation of Region 1 was induced by RdDM, C-LEC1 expression was reduced in the transgenic ECs, indicating a negative correlation between DNA methylation and C-LEC1 expression. In contrast, the hypermethylation of Region 2 did not greatly affect C-LEC1 expression. Based on these results, we hypothesize that DNA methylation may be involved in the control of C-LEC1 expression during carrot embryogenesis.