Disruption of LACCASE4 and 17 results in tissue-specific alterations to lignification of Arabidopsis thaliana stems

Abstract

Peroxidases have been shown to be involved in the polymerization of lignin precursors, but it remains unclear whether laccases (EC 1.10.3.2) participate in constitutive lignification. We addressed this issue by studying laccase T-DNA insertion mutants in Arabidopsis thaliana. We identified two genes, LAC4 and LAC17, which are strongly expressed in stems. LAC17 was mainly expressed in the interfascicular fibers, whereas LAC4 was expressed in vascular bundles and interfascicular fibers. We produced two double mutants by crossing the LAC17 (lac17) mutant with two LAC4 mutants (lac4-1 and lac4-2). The single and double mutants grew normally in greenhouse conditions. The single mutants had moderately low lignin levels, whereas the stems of lac4-1 lac17 and lac4-2 lac17 mutants had lignin contents that were 20 and 40% lower than those of the control, respectively. These lower lignin levels resulted in higher saccharification yields. Thioacidolysis revealed that disrupting LAC17 principally affected the deposition of G lignin units in the interfascicular fibers and that complementation of lac17 with LAC17 restored a normal lignin profile. This study provides evidence that both LAC4 and LAC17 contribute to the constitutive lignification of Arabidopsis stems and that LAC17 is involved in the deposition of G lignin units in fibers.

Figure 1.

Laccase Gene Expression in Arabidopsis Floral Stems (Plants Grown under Long-Day Conditions). RT-PCR expression profiles of eight laccase genes expressed in the inflorescence stem at eight different growth stages of Arabidopsis. Expression was normalized against the housekeeping β-TUBULIN (β-TUB) gene. Two signals were obtained for LAC6, corresponding to alternative splicing events. Growth stages, according to Boyes et al. (2001), are indicated at the top.

Figure 2.

Characterization of Laccase T-DNA Insertion Mutants (Grown in Long-Day Conditions). (A) Schematic diagram of the T-DNA insertion in lac4-1, lac4-2, and lac17 mutants. The positions of primers used for genotyping lac4-1 and lac4-2 and for quantitative PCR are indicated by 1, 2, and q, respectively. (B) Confirmation of the downregulation of laccase transcripts by RT-PCR. Lane 1, expression of the β-tubulin gene (housekeeping gene); lane 2, LAC4 expression; lane 3, LAC17 expression; lane M, 1 kb + ladder (Invitrogen). (C) Immunoblot analysis with an antibody against Arabidopsis LAC17. The molecular mass of Arabidopsis LAC17 is indicated by an arrow at 75 kD. (D) Quantification of laccase activity in partially purified protein extracts, with ABTS as the substrate. Data represent means ± sd (n = 3).

Figure 3.

Phenotype of Laccase Mutants. (A) Plants grown in the growth chamber (long-day conditions: no phenotype was observed). (B) Plants grown in the growth chamber (continuous light: lac4-2 lac17 displayed a semidwarf phenotype). (C) to (H) Wiesner staining of stem cross sections from plants grown under continuous light. The black arrows show collapsed xylem, and the red arrow shows hypolignified fibers. Bars = 100 μm. (I) and (J) Maüle staining of stem cross sections from wild-type and lac4-2 lac17 plants grown under continuous light. Bars = 100 μm.

Figure 4.

Analyses of Soluble Phenolic Compounds Extracted from the Stems of lac4-1, lac4-2, lac17, lac4-1 lac17, and lac4-2 lac17 Mutants (Grown in Long-Day Conditions). Data are mean values from three to nine biological replicates. Error bars indicate sd (n = 3 to 9). All results are expressed as percentages of wild-type levels (arbitrarily set at 100). SmB, sinapoyl malate in the basal part of stems; SmU, sinapoyl malate in the upper part of stems; F-Glc B, flavonol glycosides in the basal part of stems; F-Glc U, flavonol glycosides in the upper part of stems.

Figure 5.

Localization of Laccase Transcripts in Inflorescence Stem Cross Sections of GUS Promoter Fusion Lines (from Plants Grown under Long-Day Conditions). (A) Expression profile of the 2-kb ProLAC4:GUS. (B) Expression profile of the 2-kb ProLAC17:GUS. fi, fibers; vb, vascular bundle; c, cambium. Bars = 100 μm.

Figure 6.

Complementation of lac17 with Various Constructs. The effectiveness of the complementation was tested by thioacidolysis of mature stems (from plants grown under long-day conditions), assuming that lac17 mutants had stem lignins with a higher S/G thioacidolysis ratio. (A) Schematic diagram of the Gateway constructs used for lac17 complementation. (B) Impact of lac17 complementation by the endogenous promoter and genomic sequence of LAC17 without the 3′ UTR (ProLAC17:LAC17g - 3′) on the S/G ratio. (C) Impact of lac17 complementation by the endogenous promoter and genomic sequence of LAC17 with the 3′ UTR (ProLAC17:LAC17g + 3′) on the S/G ratio. The arrows indicate two lines selected for further lignin content determination on their pooled extract-free stems (lac17c17 +3′ 1; Table 6); the stems of the other two lines were pooled as lac17c17 +3′ 2 (lac17c17 +3′ 2; Table 6).