Cytokinin regulates female gametophyte development by cell cycle modulation in Arabidopsis thaliana

Male and female gametophyte development, double fertilization, and embryogenesis are key to alternating generations in angiosperms. The female gametophyte of Arabidopsis is an eight-nucleate haploid structure developed from functional megaspores (FMs) through three flawless mitoses regulated by a series of cell cycle genes. Cytokinin, an important phytohormone, plays a critical role in the regulation of plant growth and development. However, the mechanisms by which cytokinins regulate female gametophyte development remain largely unknown. In this study, we constructed transgenic plants (pES1::CKX1) with low cytokinin levels in the embryo sac. Phenotypic analysis showed that pES1::CKX1 inhibits female gametophyte development. Microscopic observation revealed that female gametophyte development of pES1::CKX1 was delayed. The promoters of all cell cycle genes were cloned and transformed into wild-type (WT). We crossed these transgenic plants of cell cycle genes expressed in ovules with pES1::CKX1 and compared the expression level of β-glucuronidase (GUS) in pES1::CKX1 and WT. Many cell cycle-regulated genes were up or downregulated in pES1::CKX1 compared with WT, and the embryo sac development cell cycle in cycd2;1/+ cycd3;3 was defective. Our results demonstrated that cytokinin affects cell division in the female gametophyte by affecting the expression of cell cycle genes.