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Isolation of Tobacco Isoperoxidases Accumulated in Cell-Suspension Culture Medium and Characterization of Activities Related to Cell Wall Metabolism1

Ario de Marco2, Patricia Guzzardi, and Élisabeth Jamet*

Institut de Biologie Moléculaire des Plantes, Unité Propre de Recherche A0406, Centre National de la Recherche Scientifique, 12 rue du Général Zimmer, 67000 Strasbourg, France

All of the most important guaiacol-type peroxidase (POX) isoforms accumulated in the culture medium of BY-2 tobacco (Nicotiana tabacum L. cv Bright Yellow 2) cells have been isolated. Five basic and two acidic isoforms were found. The four major isoforms (B2, B3, P1, and P2), all strongly basic, have been purified to homogeneity and partially sequenced. B2 and B3 are new isoforms showing high homology to only one POX isolated so far. Amino acid sequencing and specific activities indicated that basic isoPOXs constitute two pairs of strictly related isoforms (P1/P2 and B2/B3). Their specific activities measured in the presence of different substrates, as monolignols and NAD(P)H, indicated possible specialized functions in cell wall metabolism. Only P-type POXs were able to oxidize indoleacetic acid. Variations in pH could play a regulatory role by changing the relative contribution of different isoforms to total POX activity. Apart from cell culture medium, polyclonal antibodies obtained against P1 and P2 detected P1 in roots and in lower parts of stems. Immunocytochemical labeling indicated that P-type POXs were expressed in stem phloem and in phloem and epidermal cells of roots.

1   This work was supported by the Centre National de la Recherche Scientifique, by a fellowship from the Consiglio Nazionale delle Ricerche in Rome to A.d.M., and by a grant from the Ministère de l'Éducation Nationale de la Recherche et de la Technologie to P.G.
2   Present address: Novartis, Crop Protection, WRO-1060, 4002 Basel, Switzerland.
*   Corresponding author; e-mail elisabeth.jamet{at}ibmp-ulp.u-strasbg.fr; fax 33-388-614442.

Plant Physiol. (1999) 120: 371-382
Copyright Clearance Center:   0032-0889/99/120//12
© 1999 American Society of Plant Physiologists




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