Calcium and Calmodulin during Carrot Somatic Embryogenesis

Abstract

Carrot somatic embryogenesis has been used extensively as a model system for the study of control mechanisms of plant embryo-genesis. Ca2+ and the Ca2+ binding protein calmodulin play a fundamental role in the control of plant growth and development. During plant embryogenesis especially important is the role of Ca2+ in the regulation of cell polarity, cell growth, mitosis and cytokinesis, cell volume, plant hormone action and distribution, and enzyme activation. Carrot somatic embryos develop in culture on clusters of small cytoplasm rich cells. They progress through the successive stages of globular, heart shaped, and torpedo shaped embryos, comparable with zygotic embryogenesis. The process is influenced by several exogenous factors such as light quality and medium composition. Calcium and calmodulin both possess a number of chemical and physical properties which makes them very suitable as intracellular messengers in the regulation of embryogenesis. The involvement of Ca2+ and calmodulin can be studied with various techniques. Of great importance is the measurement of the cytoplasmic Ca2+ concentration and the determination of its distribution. In this paper a summary of methods for the three forms of Ca2+ (free cytoplasmic, bound to membrane surfaces or intracellular chelating molecules, and sequestered inside subcellular organelles) which are applicable to the system of carrot somatic embryogenesis, will be presented. High concentrations of external Ca2+ has proven to be promotive for carrot somatic embryogenesis. Embryogenesis increases upon transfer to a medium with a elevated concentration of Ca2+, regardless of the initial concentration. Somatic embryos possess a higher concentration of bound calcium, which is mainly localized in the outer cell layers, as compared with proembryogenic masses. Activated calmodulin is unevenly distributed in all stages of carrot somatic embryogenesis. In the globular and heart stage activated calmodulin is restricted to the basal part of the embryo. In old torpedo shaped stages activated calmodulin is also present in the shoot apex. From these observations it is concluded that Ca2+ and calmodulin are important for the initiation of polarity during carrot somatic embryogenesis.