Changes in Protein Pattern during Stress-induction of Carrot (Daucus carota L.) Somatic Embryogenesis.

Abstract

To clarify the molecular biological aspects occurring during the induction of somatic embryogenesis, the stress-induced changes in protein pattern and the proteins associated with somatic embryogenesis induced by stress in carrot (Daucus carota L.) were investigated. In two dimensional polyacrylamide gel electrophoresis (2D-PAGE), 6 spots with molecular weight of 63-65 kilo Dalton (KDa) showing pI ranging from 6.0-6.5, and 2 spots of molecular weight 45 KDa with a pI of 5.0 were detected in the extracts of apical tip segments treated with cadmium chloride, sucrose and sodium chloride. Three spots out of the 6 spots of 63-65 KDa molecular weight and the two 45 KDa spots were also detected in the extracts of 2,4-dichlorophenoxy acetic acid (2,4-D) -induced embryogenic cells, but not in those of non-embryogenic cells. Furthermore, these 5 spots were also detected in the extracts of hypocotyl segments cultured with 2,4-D for more than 4weeks. Judging from the relationship between accumulation of these proteins and acquisition of embryogenic competence, these proteins may be related to acquisition and/or maintenance of embryogenic competence.

Isolation of putative embryo-specific genes using stress induction of carrot somatic embryos

“…Many types of embryo-specific genes, including LEA-class genes, are expressed in these cells before the formation of morphologically differentiated somatic embryos (Wild et al 1988, Kiyosue et al 1991, 1993a, 1993b, Lin et al 1996, Tachikawa et al 1998. For this reason, it has been postulated that embryogenic cells are already programmed for early-and late-stage embryogenesis at the molecular level, and it is difficult to efficiently isolate early embryonic genes using these cells (Kiyosue et al 1991(Kiyosue et al , 1993a(Kiyosue et al , 1993b.…”

“…The plant materials collected were true leaves from 4-week-old plants, immature seeds from different plants at 3,15,21,27,36,45, and 54 days after flowering (DAF), and mature dry seeds. Embryogenic cells (EC) and somatic embryos were induced as described (Tachikawa et al 1998), and non-embryogenic cells (NC) that had lost the ability to form somatic embryos were used as negative controls for the acquisition of embryogenic competence (Satoh et al 1986). Murashige and Skoog (MS) media (Murashige and Skoog 1962) with or without 1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) were used as the basal media for this experiment.…”

“…Such embryos are formed directly from surface cells of the explants, without visible callus formation. In this induction system, longer stress treatments result in higher frequencies of somatic embryo formation, with stronger expression of embryo-specific genes occurring before morphological differentiation into embryos (Tachikawa et al 1998, Kikuchi et al 2006. As somatic cells of explants acquire embryogenic competence during stress treatment, and the degree of embryo formation is enhanced by increased treatment duration (Kikuchi et al 2006), some embryo-specific genes expressed in early embryogenesis are also likely to be expressed during the stress treatment, with their expression levels increasing with the duration of the stress treatment.…”

“…Carrot somatic embryos can be readily induced synchronously, in large quantities, by the transfer of embryogenic cells (EC) from auxin-containing medium to auxinfree medium (Steward et al 1958, Reinert 1959, Sung et al 1984, Kiyosue et al 1993a, 1993b (Satoh et al 1986). Several embryo-specific genes have been isolated and characterized by comparison of gene or protein expression in EC, NC, and somatic embryos (Wilde et al 1988, Kiyosue et al 1991, 1993a, 1993b, Lin et al 1996, Tachikawa et al 1998. Most of these genes were identified as LEA-class genes, and are expressed in the late stages of zygotic embryogenesis.…”

Molecular aspects of somatic-to-embryogenic transition in plants

“…Treatments such as explant wounding, or exposure to medium containing 2,4-D, may result in the rapid proliferation of cells that become undifferentiated (or de-differentiated) and lose their original polarity [219] or attain a hyperpolarized state [59]. This interference with polar auxin transport abolishes auxin gradients, which subsequently halts the programmed organogenesis and tropic responses [195,196]. This involves the erasure of existing transcriptional and translational patterns in order to redirect the developmental program of cells [42] and is marked by an increase in endogenous auxin levels [143], which along with exogenous auxin application, is a critical factor during the induction and expression of SE [81].…”