Laccase-dependent lignification of secondary cell walls of protoxylem tracheary elements in Arabidopsis thaliana

Lignin is a phenolic polymer that plays important roles in the structural integrity of plants. Both peroxidases and laccases have been implicated in the polymerization of lignin, and mutant analyses have conclusively demonstrated a role for laccases in lignification of Arabidopsis thaliana stems. However, the oxidative enzymes that polymerize lignin in protoxylem tracheary elements (TEs) have not been defined. Induction of the master transcription factor VASCULAR RELATED NAC-DOMAIN 7 (VND7) causes systemic transdifferentiation into protoxylem TEs, providing an inducible-experimental model system to study protoxylem TE differentiation. The transcriptome of these lines has been well characterized, and two laccases, LAC4 and LAC17, are strongly expressed following induction of protoxylem TE development. To test if LAC4 and LAC17 are necessary for the lignification of protoxylem TEs, the inducible VND7 construct was transformed into the lac4-2/lac17 double mutant background and fluorescently labeled monolignols were exogenously applied to differentiating protoxylem TEs. Labeled polymerized lignin was only detected in the wild-type protoxylem TEs, but not in lac4-2/lac17 protoxylem TEs. To test if laccases alone are sufficient to promote lignification, the constitutive 35S promoter was used to drive either LAC4 or LAC17 in wild-type plants, resulting in strong ectopic lignification of primary cell walls upon application of fluorescently labeled monolignols. Fluorescently tagged laccases were transformed into the inducible protoxylem TEs system, where they specifically localize to the secondary, but not primary, cell walls of protoxylem tracheary elements. This research shows that LAC4 and LAC17 are necessary and sufficient for the lignification of secondary cell wall domains of protoxylem TEs and that they are specifically localized to these domains.Science, Faculty ofBotany, Department ofGraduat