Development of methodology for freeze-substitution and immunolocalization in differentiating tracheary elements of Zinnia elegans

Abstract

In higher plants, redifferentiation occurs when one cell type transforms into another with an associated change in cell function. Xylem tissue is formed either in this manner, from parenchyma cells, or by de novo differentiation from procambium/cambium initials. Tracheary elements (TEs) are water-conducting cells in xylem tissue. Dead at maturity, these elements connect end to end to form a transport system In culture, parenchyma cells have been shown to redifferentiate into tracheary elements, forming secondary wall patterns by locahzed thickenings (Kohlenbach et al., 1981) (see Figs. 1.1 and 1.2). Zinnia elegans has become a model system by which to study TE differentiation because single cells can be induced to form TEs in culture.

Present in only specialized cell types such as TEs, the secondary cell wall is laid down inside the primary ceU waU under cytoskeletal control. The control of secondary wall development is not well defined. Actin and microtubules are two cytoskeletal elements that theory and observations predict will be involved in this process. Research with antibodies in the Zinnia system can reveal whether or not any pattem is recognizeable before the secondary wall thickenings appear and the morphological stages can then be identified that represent the estabhshment of that pattem. "The possible role of cytoskeletal elements in regulating microfibril organization remains one of the central unresolved questions in plant cell biology... diflSculty arises in proving that the cytoskeleton is essential for the organized deposition of wall microfibrils" (Seagull 1989. p. 145). The focus of this research is to optimize uhra rapid freezing methods so that routine experiments performed on Zinnia TEs will resuh in state-of-the-art preservation of cytoplasm and immuno-gold labeUng by antibodies.