Molecular cloning and expression of eight laccase cDNAs in loblolly pine (Pinus taeda)

Eight cDNA clones for laccase (Lac 1-Lac 8) were isolated and characterized for analysis of the role of laccases in xylem formation by molecular and genetic approaches. Five of them (Lac 1-5) were isolated from cDNA libraries derived from differentiating xylem through a cDNA sequencing project in loblolly pine xylem (Allona of al. 1998) and other three (Lac 6-8) were isolated using a probe from a tobacco blue copper oxidase. Analysis of the cDNAs revealed that the proteins predicted had N-terminal signal sequences, 8-21 glycosylation sites, and four copper binding sites. The putative mature laccases range between 59.2 and 61.7 kD. The predicted isoelectric points vary between 7.3 and 9.9. Phylogenetic analysis shows these laccase cDNAs form three clusters in the cationic plant laccase group. Northern analysis indicated that all eight of the laccase transcripts are most abundant in differentiating xylem of the six tissues and organs tested. The Lad transcript is also detectable in immature pollen cones, and the Lac 7 transcript is detectable in several organs tested. The predominance of transcript abundance in differentiating xylem suggests that laccases play an important role in pine xylem development, consistent with previous hypotheses that laccases are involved in lignin synthesis in differentiating xylem.