Imaging of polarity during zygotic and somatic embryogenesis of carrot (Daucus carota L.)

In this thesis a study of the regulation of coordinated growth and the development of polarity during embryogenesis of carrot, Daucus carota L. , is described. To this end, several microscopical techniques were used, such as light microscopy, fluorescence microscopy, confocal scanning laser microscopy and electron microscopy. Next to this, immunocytochemical methods were used frequently to localize proteins in plant tissue sections.Plants are composed of several types of organs and tissues, each of them having a characteristic structure and function. For the development of a full-grown germling from one cell, the zygote, a tight regulation of growth and differentiation is required. During this process of embryogenesis, growth proceeds through a number of developmental stages which are described subsequently as globular, oblong, heartshaped and torpedo-shaped.Despite the large number of observations on embryogenesis, made in various plants, the molecular and cellular basis of this developmental pathway is still poorly understood. The divalent cation Ca 2+ participates in the initiation and maintenance of a great variety of physiological processes, including the regulation of cell polarity, cell division, cell growth, cell volume, hormone action and distribution, and enzyme synthesis and activation. Considering the diversity of processes in which Ca 2+ is involved, it is to be expected that an investigation of the distribution of Ca 2+ , and Ca 2+ binding proteins, during plant embryogenesis, will lead to a deeper understanding of the regulation of this process.Studies on zygotic embryogenesis are hampered by the presence of surrounding maternal tissue. Therefore, somatic embryos of carrot are used often as experimental substitutes for zygotic embryos, since the discovery of in vitro embryogenesis in carrot cultures in 1958. Carrot somatic embryos can be obtained, relatively easily, in great amounts, essentially free of surrounding tissue, just by transferring cell clusters, designated as proembryogenic masses, from medium supplemented with the growth regulator 2,4-D to medium without 2,4-D. This feature makes carrot an ideal model system for the study of plant embryogenesis.In Chapter 1, the general introduction, the zygotic and somatic embryogenesis of carrot is described structurally. Similarities and differences between both processes are mentioned. Many external factors, which are described extensively in the literature, influence the development of somatic embryos. For normal growth and development to occur, the presence of Ca 2+ in the medium is absolutely required, and embryogenesis is enhanced specifically by a rise of [Ca 2+ ]. In this chapter, the role and distribution of Ca 2+ in plants is briefly described.The principal targets of calcium signals in eukaryotes are calciumbinding proteins of which calmodulin, a protein present in all plant cells, has been studied most extensively. The structure, activity and localization of this acidic, small and heatresistant protein is described from the literature. Chapter 1 ends with a survey of techniques which are nowadays available for the localization of Ca 2+ and calmodulin in plants.In Chapter 2, chlorotetracycline and fluphenazine, two fluorescent indicators, are being used to localize Ca 2+ and activated calmodulin respectively, during carrot somatic embryogenesis. Embryogenesis appears to coincide with a rise in [Ca 2+ ] and activated calmodulin is mainly found in the future root side of the embryo. It is concluded, that the polarity in the distribution of calmodulin is already present before polarity is visible morphologically.Fluphenazine visualizes only activated calmodulin. In Chapter 3, the distribution of both activated and non- activated calmodulin has been studied with the aid of antibodies. Besides the various developmental stages of somatic embryogenesis, also zygotic embryos and a number of stages of zygotic embryo germination have been studied in this chapter. The most striking observation is that the distribution of calmodulin in somatic embryos differs strongly from the distribution in zygotic embryos, but resembles the distribution during zygotic embryo germination. Both in somatic embryos and in germinated zygotic embryos, calmodulin appears to be present mainly in amyloplasts, while in zygotic embryos calmodulin predominantly was found to be localized in the cytoplasm.For a detailed analysis of the distribution of Ca 2+ in living, intact plant cells in tissues with fluorescent indicators, confocal laser scanning microscopy is the method of choice. A suitable indicator is fluo-3. Unfortunately, the plasma membrane is not permeable for this compound. Therefore, a method had to be developed with which fluo-3 could easily be loaded into plant cells. In Chapter 4, it is described that with the aid of digitonin fluo-3 can be used successfully for the localization of Ca 2+ in embryogenic plant cells, in combination with confocal scanning laser microscopy.As has already been noticed in Chapter 2, carrot somatic embryogenesis coincides with a rise in [Ca 2+ ]. In Chapter 5, a detailed analysis has been made of the distribution of free cytosolic Ca 2+ during carrot somatic embryogenesis with the aid of the method described in Chapter 4. It appeared that [Ca 2+ ] is especially high in the protoderm of the embryos, and gradients in [Ca 2+ ] along the longitudinal axis of torpedo-shaped embryos were frequently observed. Very obvious was the high [Ca 2+ ] in the nuclei of protoderm cells, This nuclear localization was confirmed by antimonate precipitation, by which Ca 2+ is visualized as electron dense precipitates in the electron microscope.The concentration of Ca 2+ in the cytosol is not only linked with the concentration in the vacuole, but also with the pH of the cytosol and the vacuole. Since both pH and [Ca 2+ ] are important factors during embryo genesis, in Chapter 6 a study is described of the distribution of pH in vacuoles during somatic and zygotic embryogenesis and during zygotic embryo germination of carrot. Neutral red and acridine orange were used as indicators of vacuolar pH and their distribution has been compared with the distribution of fluphenazine. Strikingly, major similarities were found between the distribution of the three probes used, and all three reacted similarly on treatments with A231 87, EGTA or propionic acid. Confocal microscopy revealed a network of vesicles and tubules, predominantly present in the protoderm of somatic embryos and germinated zygotic embryos after incubation in acridine orange. Proposed is, that calmodulin is possibly involved in the digestion of cell material in autophagic vacuoles or is involved in the regulation of the movements of vacuolar tubules. However, additional research is necessary to explain the observed distribution patterns satisfactory.From the previous chapters it appeared that noticeable differences exist between somatic and zygotic embryos in the distribution of anti calmodulin, fluphenazine fluorescence, neutral red and acridine orange. In Chapter 7 a possible structural basis of these differences has been searched for. The distribution of anti-calmodulin, and perhaps also fluphenazine fluorescence, could be linked with the presence of amyloplasts, which were abundant in somatic and germinated zygotic embryos, but which were not found in zygotic embryos. Differences in the localization of neutral red and acridine orange are related to differences in vacuolation between somatic and zygotic embryos.Chapter 8 is the general discussion. Here, somatic embryo initiation, its early development and the formation of polarity during somatic and zygotic embryogenesis of carrot are the main topics. It is concluded, that important differences exist between somatic and zygotic embryogenesis and that the process of somatic embryogenesis shows similarities with zygotic embryo germination. The chapter ends with some concluding remarks about the methods used for the localization of Ca 2+ and calmodulin.