Isolation of tobacco isoperoxidases accumulated in cell-suspension culturemedium and characterization of activities related to cell wall metabolism

All of the most important guaiacol-type peroxidase (POX) isoforms accumulat ed in the culture medium of BY-2 tobacco (Nicotiana tabacum L. cv Bright Ye llow 2) cells have been isolated. Five basic and two acidic isoforms were f ound. The four major isoforms (B2, B3, P1, and P2), all strongly basic, hav e been purified to homogeneity and partially sequenced. B2 and B3 are new i soforms showing high homology to only one POX isolated so far. Amino acid s equencing and specific activities indicated that basic isoPOXs constitute t wo pairs of strictly related isoforms (P1/P2 and B2/B3). Their specific act ivities measured in the presence of different substrates, as monolignols an d 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 diff erent isoforms to total POX activity. Apart from cell culture medium, polyc lonal antibodies obtained against P1 and P2 detected P1 in roots and in low er parts of stems. Immunocytochemical labeling indicated that P-type POXs w ere expressed in stem phloem and in phloem and epidermal cells of roots.