Molecular characterization of glycoproteins secreted by carrot suspension cells

<p>Investigations into the molecular mechanisms that underly plant embryogenesis suffer from the limited accessibility of the developing zygotic embryo, which is enveloped by the ovary in the flower and, later on, by the developing seed. The formation of embryos from cultured somatic cells, referred to as somatic embryogenesis, provides an alternative means to study embryo development. In carrot cell suspension cultures, somatic embryogenesis is highly efficient and embryos at different developmental stages can be easily obtained in high quantities. For this reason, carrot cell suspension cultures are often used as an alternative to investigate plant embryogenesis.<p>The growth medium of plant cell cultures can be regarded as a large extension of the intercellular space; soluble secreted molecules that inhabit the apoplast <em>in planta</em> will <em></em> accumulate in the medium of suspension-cultured cells. Cultured carrot cells secrete many proteins, most of which are glycosylated, into the growth medium. A correlation was found between somatic embryogenesis and the presence or absence of some of these secreted proteins. Evidence was obtained that one or more. secreted glycoproteins are actually essential for somatic embryo formation.<p>Apart from embryogenic cells, carrot cell suspensions contain non-embryogenic cells that contribute to the spectrum of proteins in the culture medium. These proteins may interfere with somatic embryo development or with studies that further analyse the role of extracellular proteins in somatic embryogenesis.<p>The aim of the study presented in this thesis was to increase our knowledge of the nature and action of glycoproteins secreted by carrot suspension cells and of the cell type-specific expression of the encoding genes both in suspension cultures and <em>in planta.</em><p>In chapter 1 a brief introduction in zygotic and somatic embryogenesisis is presented, and the current evidence for the involvement of secreted glycoproteins in carrot somatic embryogenesis is summarized. Chapters 2, 3 and 4 describe the molecular characterization of the carrot secreted glycoproteins EP1 and EP4. Antisera specific for EP1 and EP4 and cDNA clones encoding these proteins were employed to study their occurrence in cell suspensions and in seedlings. The results show that the morphological cell-type heterogeneity in carrot cell suspension cultures is parallelled by differences in secreted proteins and suggest that this heterogeneity reflects the different cell types of the explant. Both the EP1 and EP4 proteins were purified from the culture medium and tested for their effect on somatic embryo formation. These experiments revealed neither a promoting nor an inhibiting role for EP1 or EP4 in somatic embryogenesis. In chapter 5 the current knowledge on the structure and function of the plant primary cell wall is reviewed. In this context, the involvement of secreted glycoproteins in somatic embryogenesis and the possible functions of EP1 and EP4 are discussed