Immunocytochemical localization of phenylalanine ammonia-lyase and cinnamyl alcohol dehydrogenase in differentiating tracheary elements derived from Zinnia mesophyll cells

Secondary wall thickening is the most characteristic morphological feature of the differentiation of tracheary elements. Isolated mesophyll cells of Zinnia elegans L. cv. Canary Bird in differentiation medium are converted to tracheary elements, which develop lignified secondary wall thickenings. Using this system, we investigated the distribution of two enzymes, phenylalanine ammonia-lyase (PAL) (EC 4.3.1.5) and cinnamyl alcohol dehydrogenase (CAD) (EC 1.1.1.195), by both biochemical and immunological methods. Both PAL and CAD appear to be key enzymes in the biosynthesis of lignin precursors, and they have been shown to be associated with the differentiation of tracheary elements. Cultured cells were collected after various times in culture. The culture medium was separated from cells by centrifugation and designated fraction (1), the extracellular fraction. The collected cells were homogenized and separated into four fractions: (2) cytosol; (3) microsomes; (4) cell walls (loosely bound material); and (5) cell walls (tightly bound material). PAL activity was detected in each fraction. The extracellular fraction consistently had the greatest PAL activity. Moreover, PAL activity in the cytosolic fraction increased rapidly prior to lignification, as it did in both the microsomal and the cell wall (tightly bound) fractions during lignification. Antisera against PAL and against CAD detected the proteins with molecular masses that corresponded to those of PAL and CAD in Zinnia. Immuno-electron microscopy revealed that, in differentiating tracheary elements, PAL was dispersed in the cytoplasmic matrix and was located on Golgi-derived vesicles and on the secondary wall thickenings. "Cell-free" immuno-light microscopy supported the putative distribution of PAL on lignifying secondary walls. The pattern of distribution of CAD was similar to that of PAL. Thus, both PAL and CAD seemed to be localized in secondary wall thickenings. From the results of both biochemical assays and immunocytochemical staining, it appeared that at least two types of PAL and CAD are present in differentiating cells. One type of each enzyme is distributed in the cytosol, while the other is secreted from the Golgi apparatus and transported by Golgi-derived vesicles to the secondary wall thickenings.