| Summary: | 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 of === Botany, Department of === Graduate
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