PHYSIOLOGIA PLANTARUM 114: 343–353. 2002 Copyright C Physiologia Plantarum 2002 Printed in Denmark – all rights reserved ISSN 0031-9317 Lignification related enzymes in Picea abies suspension cultures Anna Kärkönen a,* , Sanna Koutaniemi b , Maaret Mustonen a , Kaisa Syrjänen c , Gösta Brunow c , Ilkka Kilpeläinen b , Teemu H. Teeri b , Liisa Kaarina Simola a a Department of Biosciences, Division of Plant Physiology, PO Box 56, FIN-00014 University of Helsinki, Finland b Institute of Biotechnology, PO Box 56, FIN-00014 University of Helsinki, Finland c Laboratory of Organic Chemistry, Department of Chemistry, PO Box 55, FIN-00014 University of Helsinki, Finland * Corresponding author, e-mail: anna.karkonen/helsinki.fi Received 11 May 2001; revised 24 September 2001 Activity of a number of enzymes related to lignin formation was measured in a Picea abies (L) Karsten suspension culture that is able to produce native-like lignin into the nutrient me- dium. This cell culture is an attractive model for studying lignin formation, as the process takes place independently of the complex macromolecular matrix of the native apoplast. Suspension culture proteins were fractionated into soluble cel- lular proteins, ionically and covalently bound cell wall proteins and nutrient medium proteins. The nutrient medium contained up to 5.3% of total coniferyl alcohol peroxidase (EC 1.11.1.7) activity and a significant NADH oxidase activity that is sug- gested to be responsible for hydrogen peroxide (H 2 O 2 ) pro- duction. There also existed some malate dehydrogenase (EC 1.1.1.37) activity in the apoplast of suspension culture cells (in ionically and covalently bound cell wall protein fractions), possibly for the regeneration of NADH that is needed for per- oxidase-catalysed H 2 O 2 production. However, there is no proof of the existence of NADH in the apoplast. Nutrient medium peroxidases could be classified into acidic, slightly Introduction Lignin is an integral cell wall component of all vascular plants. The biosynthetic pathway to the lignin precur- sors, monolignols, is quite well known (for a review see Lewis et al. 1999). Lignin polymerization in higher plants proceeds initially through the dehydrogenation of substituted p-hydroxycinnamyl alcohols and through later coupling of the phenol radicals to give the lignin polymer. However, the identity of the enzyme(s) catalys- ing the oxidative polymerization of monolignols during lignin synthesis is a matter of controversy. Classical se- cretory plant peroxidases (class III; EC 1.11.1.7; donor: Abbreviations –– ABTS, 2,2ƒ-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid); CAO, coniferyl alcohol oxidase; DAB, 3,3ƒ-diaminobenzidine; DHP, dehydrogenation polymer; HPLC, high pressure liquid chromatography; IEF, isoelectric focusing; LC-MS, liquid chromatography – mass spectrometry; MDH, malate dehydrogenase; NMR, nuclear magnetic resonance; 4-NPG, 4-nitrophenyl b-glucopyranoside; PMSF, phenylmethylsulphonyl fluoride. Physiol. Plant. 114, 2002 343 basic and highly basic isoenzyme groups by isoelectric focus- ing. Only acidic peroxidases were found in the covalently bound cell wall protein fraction. Several peroxidase isoen- zymes across the whole pI range were detected in the protein fraction ionically bound to cell walls and in the soluble cellu- lar protein fraction. One laccase-like isoenzyme with pI of approximately 8.5 was found in the nutrient medium that was able to form dehydrogenation polymer from coniferyl alcohol in the absence of H 2 O 2 . The total activity of this oxidase towards coniferyl alcohol was, however, several orders of mag- nitude smaller than that of peroxidases in vitro. According to 2D 1 H- 13 C correlation NMR spectra, most of the abundant structural units of native lignin and released suspension cul- ture lignin are present in the oxidase produced dehydrogen- ation polymer but in somewhat different amounts compared to peroxidase derived synthetic lignin preparations. A coni- ferin b-glucosidase (EC 3.2.1.21) was observed to be secreted into the culture medium. hydrogen peroxide oxidoreductase) are a group of most- ly N-glycosylated, heme-containing monomeric enzymes that can oxidize a large variety of substrates mainly by using H 2 O 2 as oxidizing agent (Welinder 1992). Laccases (p-diphenol : oxygen oxidoreductase, EC 1.10.3.2) are O 2 -dependent, blue-copper phenoloxidases containing four copper atoms in the active site. They are glycosylat- ed, monomeric enzymes (Dean and Eriksson 1994). Both peroxidases and laccases, or their combination, have been proposed to be involved in lignin biosynthesis. Relationships between peroxidase isoenzymes and the