ARCHIVES OFBIOCHEMISTRY AND BIOPHYSICS Vol. 269, No. 2, March, pp. 455-462,1989 Formation of trans-Caff eoyl-CoA from trans-4-Coumaroyl-CoA by Zn2+-Dependent Enzymes in Cultured Plant Cells and Its Activation by an Elicitor-Induced pH Shift RICHARD E. KNEUSEL,* ULRICH MATERN,*,’ AND KLAAS NICOLAYt *Biologisches Institut II der Universitiit Freiburg, Biochemie der Pjanzen, Schknzlestrasse 1, D-7800 Freiburg, Federal Republic of Germany; and tRijksuniversiteit te Utrecht, Instituut VOW- Mokculaire Biologie en Medische Biotechnologie, Padualaan 8, Postbus 80.063,3508 TB Utrecht, The Netherlands Received July 15,1988, and in revised form October 1,1988 A novel hydroxylase activity catalyzing the formation of trans-caffeoyl-CoA from trans-4-coumaroyl-CoA was identified in crude extracts from cultured parsley cells. The extracts were less active (V,,,,,/K,) in converting trans-4-coumaric to bans-caffeic acid. Optimal hydroxylase activity was found at pH 6.5 with a steep decline toward both pH 7.4 and pH 5.0. The enzyme activity requires ascorbate and Zn2+ at optimal concentra- tions of 50 and 0.5 mM, respectively. No other reductant could replace ascorbate, whereas high concentrations of Ca2+ partially substituted for Zn2+. The enzyme is soluble and appears to be located in the cytoplasm. The unusual pH optimum suggests that the hy- droxylase is inactive at the normal cytoplasmic pH. Upon treatment of parsley cells with an elicitor derived from Phytophthoru megasperma f. sp. glycinea, the cytoplasmic pH dropped by approximately 0.25 pH unit within 55 min as determined by 31P NMR spec- troscopy. Our results suggest that this shift in the cytoplasmic pH is sufficient for the activation of the hydroxylase, eventually leading to the formation of caffeoyl and feru- loyl esters. Such esters may be a part of a very rapid resistance response of the plant cells, which would leave no time for de novo enzyme synthesis. (c: 1989 Academic press. me. A number of factors play an important role in a plant’s defense against potential pathogens. Among these are preformed physical and chemical barriers such as cu- tin or phenolic substances. On the other hand, plant resistance may also be the re- sult of induced defense mechanisms in which metabolic processes in the plant cells are drastically changed upon contact with the potential pathogen (1). Known in- duced defense mechanisms include cell wall modifications due to the synthesis of callose and phenolic complexes such as lig- nin or suberin, the accumulation of hy- droxyproline-rich proteins in the cell wall, and the accumulation and excretion of ’ To whom correspondence should be addressed. phytoalexins (2-6). The activation of most disease resistance mechanisms is thought to be due primarily to the transcriptional induction of the relevant genes (7-10). However, it has been shown that the p-1,3- glucan synthase responsible for callose biosynthesis, for example, is activated by a local increase in cytoplasmic Ca2+ concen- tration (11). Other intracellular factors such as shifts in pH (12) and the distribu- tion of inorganic phosphate (13) have also been suggested as being involved in the in- duction of defense responses. The hydroxylation of 4-coumaric acid to caffeic acid is a central reaction in the syn- thesis of lignin and other phenolic sub- stances. The catalysis of this reaction has often been ascribed to a phenolase enzyme which requires molecular oxygen and a re- 455 0003-9861/89 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.