Membrane lipid raft organization during cotton fiber development

Background: Cotton fiber is a single-celled seed trichome that originates from the ovule epidermis. It is an excellent model for studying cell elongation. Along with the elongation of cotton fiber cell, the plasma membrane is also extremely expanded. Despite progress in understanding cotton fiber cell elongation, knowledge regarding the relationship of plasma membrane in cotton fiber cell development remains elusive. Methods: The plasma membrane of cotton fiber cells was marked with a low toxic fluorescent dye, di-4-ANEPPDHQ, at different stages of development. Fluorescence images were obtained using a confocal laser scanning microscopy. Subsequently, we investigated the relationship between lipid raft activity and cotton fiber development by calculating generalized polarization (GP values) and dual-channel ratio imaging. Results: We demonstrated that the optimum dyeing conditions were treatment with 3 mu mol.L-1 di-4-ANEPPDHQ for 5 min at room temperature, and the optimal fluorescence images were obtained with 488 nm excitation and 500-580 nm and 620-720 nm dual channel emission. First, we examined lipid raft organization in the course of fiber development. The GP values were high in the fiber elongation stage (5-10 DPA, days past anthesis) and relatively low in the initial (0 DPA), secondary cell wall synthesis (20 DPA), and stable synthesis (30 DPA) stages. The GP value peaked in the 10 DPA fiber, and the value in 30 DPA fiber was the lowest. Furthermore, we examined the differences in lipid raft activity in fiber cells between the short fiber cotton mutant, Li-1, and its wild-type. The GP values of the Li-1 mutant fiber were lower than those of the wild type fiber at the elongation stage, and the GP values of 10 DPA fibers were lower than those of 5 DPA fibers in the Li-1 mutant. Conclusions: We established a system for examining membrane lipid raft activity in cotton fiber cells. We verified that lipid raft activity exhibited a low-high-low change regularity during the development of cotton fiber cell, and the pattern was disrupted in the short lint fiber Li-1 mutant, suggesting that membrane lipid order and lipid raft activity are closely linked to fiber cell development.