Lignin synthesis and its related enzymes as markers of tracheary-element differentiation in single cells isolated from the mesophyll of Zinnia elegans

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Abstract
Mesophyll cells isolated from Zinnia elegans L. cv. Canary Bird were cultured for 96 h in a liquid medium containing 0.1 mg l-1 α-naphthaleneacetic acid and 1 mg l-1 benzyladenine in which both differentiation of tracheary elements (TE) and cell division were induced, or in a medium containing 0.1 mg l-1 α-naphthaleneacetic acid and 0.001 mg l-1 benzyladenine, in which cell division was induced but TE differentiation was not. Lignification was found to occur only in the former medium, fairly synchronously after 76 h of culture, 5 h later than the onset of visible secondary wall thickening. Changes in the soluble phenolics were not correlated with TE differentiation. Of three important enzymes which have been reported to play a role in TE differentiation, the activity of phenylalanine ammonia-lyase (EC 4.3.1.5) in the TE-inductive culture was higher than that in the control culture between 72 and 96 h of culture, when TE differentiation progressed and lignin was synthesized actively. O-Methyltransferase (EC 2.1.1.6) activity was higher in the control culture than in the TE-inductive culture, indicating that this enzyme was not a marker enzyme of TE differentiation. The activities of peroxidases (EC 1.11.1.7), one extractable and the other nonextractable, with CaCl2 from the cell walls, reached peaks at 72 h (just before lignification) and 84 h of culture (active lignin synthesis), respectively, in the TE-inductive culture only, whereas the activity of soluble peroxidase showed a similar pattern of increase in the TE-inductive to the control culture. These results indicate that phenylalanine ammonia-lyase and peroxidase bound to the cell walls can be marker proteins for the differentiation of TE.