Characterization of a cationic cell-wall-bound peroxidase responsible for the dehydrogenative polymerization of lignin in Populus alba L

Previously we reported that purified CWPO-C (Cell Wall Peroxidase-Cationic) from poplar callus (Populus alba L.) showed a strong substrate preference for sinapyl alcohol and oxidizes polymeric substrate unlike other plant peroxidases and proposed that this isoenzyme is a conceivable lignification specific peroxidase. In this study, the cDNA encoding CWPO-C (ORF 972 bp) was isolated by the reverse transcriptase chain reaction technique using the specific primers designed from the partial amino acid sequences of CWPO-C. The deduced CWPO-C amino acid sequence showed that all the key residues of the plant peroxidase active site and Cys residues are maintained in CWPO-C protein. We investigated the localization of CWPO-C protein in the differentiating xylem of poplar stem. Immunohistochemical analysis showed that CWPO-C located middle lamellae, cell comer, and secondary cell walls of the fiber cells that are lignifying and mature stage during the secondary wall formation. CWPO-C labeling increased gradually from cell wall thickening stage to mature stage of fiber, that is very consistent with the increase of lignin content in the developing xylem. These results also strongly support that CWPO-C is responsible for the lignification of the secondary xylem. The CWPO-C signals kept increase in the middle lamellae, cell comers, and secondary walls even after the programmed death of the fiber cells. Instead, the signals were detected within the parenchyma cells in the mature stage of wall formation. The literatures suggested that enzymes involved in the monolignol biosynthesis also distributed in the parenchyma cells. These observations suggest that lignification of the secondary xylem proceeds even after the programmed cell death by the supply of monolignols and lignification peroxidase from parenchyma cells.