The carrot extracellular lipid transfer protein EP2

Abstract

In many plant species embryos can develop from cultured somatic cells in a process referred to as somatic embryogenesis. Apart from their cellular origin somatic embryos develop through the same characteristic morphological stages, i.e. globular, heart and torpedo stages, observed during zygotic embryogenesis. Somatic embryos of different developmental stages can easily be obtained in large quantities, one of the main reasons for the frequent use of somatic embryogenesis as an alternative system to study plant embryogenesis.Despite the wealth of information available on both zygotic and somatic embryo development with respect to their morphology and histology, insight in the molecular events that take place during embryogenesis is still poor. The study presented in this thesis is based on the observation that the presence of a small number of (glyco)proteins secreted into media of cultured carrot cells parallels the appearance of actual somatic embryos in these cultures. The aim of this study was to clone genes encoding these proteins and to study their expression and function in relation to embryogenesis. The approach taken was to screen an expression library with antisera raised against proteins secreted by carrot cells. This screening resulted in four cDNA clones which, upon detailed analysis of the expression patterns of the corresponding genes and identification of the respective encoded proteins by immunological means, were shown to represent genes encoding proteins secreted by distinct cell types.In chapter 1 an introduction to both somatic and zygotic embryogenesis is presented, as well as an overview and classification of genes and proteins expressed in embryos that have been identified in other studies, based on their temporal expression patterns during embryo development. Chapter 2 describes the cloning of four cDNAs representing the carrot genes Extracellular Protein (EP)1, EP2, EP5 and EP6. Based on the observed close correlation of EP2 gene expression with embryo development, this gene was chosen for further study as described in chapter 3. In situ hybridization experiments revealed EP2 expression already in the 60-celled zygotic embryo, and in proembryogenic masses, the precursors of somatic embryos, and in somatic embryos. Based on its sequence homology with other extracellular non-specific lipid transfer proteins, the expression of the EP2 gene in protodermal and epidermal cells in embryos and plants, and the known coincidence of a cuticle on those cells, a role for the EP2 encoded protein was proposed in the transport of monomeric cutin precursors to sites of cutin synthesis. To support this hypothesis, the affinity of the purified EP2 protein for known cutin precursors was demonstrated in chapter 4. In chapter 5 the existing approaches to study early plant embryo development are discussed and compared with the results presented here