Spatial and temporal regulation of lignification during tracheary element differentiation

During tracheary element (TE) differentiation, lignin is deposited specifically to secondary cell walls. Spatial and temporal regulation of lignification during TE differentiation was investigated using an experimental system in which TEs are differentiated from isolated Zinnia mesophyll cells. The mechanism how and whence monolignols are supplied to TEs undergoing programmed cell death was investigated. Analysis by HPLC and GC-MS showed that coniferyl alcohol, coniferaldehyde, and sinapyl alcohol were accumulated in cultured medium during differentiation inductive culture. The concentration of coniferyl alcohol peaked at the beginning of secondary wall thickening, decreased rapidly during secondary wall formation, then increased again. These results indicated that lignification of TEs progresses by supply of monolignols from not only TEs themselves but also surrounding xylem parenchyma-like cells through medium in vitro. Simultaneously, these results would suggest that lignification of TEs in vivo progresses by supply of monolignols from not only TEs themselves but also surrounding xylem parenchyma cells through apoplast. For study of the final step of lignification, polymerization of monolignols, cell wall-bound peroxidase isoenzymes were analyzed, and a cationic isoenzyme P5 was shown to appear specifically for cells differentiating into TEs. Characterization of P5 strongly suggested that P5 is involved in lignin biosynthesis during TE differentiation. Furthermore, a peroxidase gene, ZPO-C, was isolated by PCR method. Transcripts of ZPO-C were accumulated transiently during thickening of secondary walls of TEs. By immunoelectron microscopy, the ZPO-C protein was shown to localize specifically in the lignified parts of secondary walls of TEs. On the other hand, basic laccases appeared specifically in differentiation inductive culture, too. In conclusion, it was shown that the monolignols would be supplied to TEs from other cells, and polymerized to lignin by the peroxidases and/or laccases localized specifically in secondary cell walls of TEs during TE differentiation of cultured Zinnia mesophyll cells.