Immunohistochemical localization of enzymes that catalyze the long sequential pathways of lignin biosynthesis during differentiation of secondary xylem tissues of hybrid aspen (Populus sieboldii x Populus grandidentata)

We have investigated the spatial localization of enzymes that catalyze the sequential pathways of lignin biosynthesis in developing secondary xylem tissues of hybrid aspen (Populus sieboldii Miq. x Populus grandidentata Michx.) using immunohistochemical techniques. The enzymes phenylalanine ammonia-lyase, caffeic acid 3-O-methyltransferase and 4-coumarate:CoA ligase in the common phenylpropanoid pathway, cinnamyl-alcohol dehydrogenase (CAD) and peroxidase in the specific lignin pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) in the shikimate pathway and glutamine synthetase (GS) in the nitrogen reassimilation system were abundantly localized in the 6th to 9th wood fibers away from cambium; these wood fibers are likely undergoing the most intense lignification. Only weak immunolabeling of enzymes involved in the general phenylpropanoid and specific lignin pathways was detected in the cells near the cambium; lignification of these cells has likely been initiated after primary cell wall formation. In contrast, distinct localization of DAHPS and GS was observed around the cambium, which may be involved not only in lignin biosynthesis, but also in amino acid and protein synthesis, which are essential for cell survival. Our observations suggest that co-localization of enzymes related to the sequential shikimate, general phenylpropanoid and specific lignin branch pathways and to the nitrogen recycling system is associated with cell wall lignification of wood fibers during secondary xylem development.