In situ detection of laccase activity and immunolocalisation of a compression-wood-specific laccase (CoLac1) in differentiating xylem of Chamaecyparis obtusa

Abstract

The secondary cell wall of compression wood tracheids has a highly lignified region (S2 L) in its outermost portion. To better understand the mechanism of S2 L formation, we focussed on the activity of laccase (a monolignol oxidase) and performed in situ studies of this enzyme in differentiating compression wood. Staining of differentiating compression wood demonstrated that laccase activity began in all cell wall layers before the onset of lignification. We detected no activity of peroxidase (another monolignol oxidase) in any cell wall layer. Thus, laccase likely plays the major role in monolignol oxidisation during compression wood differentiation. Laccase activity was higher in the S2 L region than in other secondary wall regions, suggesting that this enzyme was responsible for the high lignin concentration in this region of the cell wall. Immunolabelling demonstrated the expression of a compression-wood-specific laccase (CoLac1) immediately following the onset of secondary wall thickening, this enzyme was localised to the S2 L region, whereas much less abundant in the S1 layer or inner S2 layer. Thus, the CoLac1 protein is most likely localised to the outer part of S2 and responsible for the high lignin concentration in the S2 L region.

Difference in enzymatic dehydrogenative polymerization of dilignols using horseradish peroxidase and crude enzyme obtained from Japanese cypress (Chamaecyparis obtusa)

“…The crude enzyme was isolated using the method reported by Hiraide et al [15]. After debarking, the differentiating xylem was scraped off using a peeler and frozen by immersing in liquid nitrogen.…”

Common Mechanism of Lignification of Compression Wood in Conifers and <i>Buxus</i>

“…To analyze in situ laccase activity in differentiating xylem, we performed laccase activity staining using 3,3'diaminobenzidine tetrahydrochloride (DAB) [17]. One of the twin blocks (see plant materials) was treated with catalase in sodium acetate-acetic acid buffer (pH 5.0) for 30 min to remove H 2 O 2 , preventing peroxidase activity.…”

“…Asterisks indicate cells that have begun secondary wall thickening (confirmed by polarizing microscopy [20] [21]). In addition, to investigate in situ laccase activity in differentiating reaction wood, we performed laccase activity staining (Figure 3(A), Figure 3(B), Figure 3(D), Figure 3(F)), in which a brown color shows laccase activity [17]. The negative control was autoclaved to inactivate laccase activity; no staining was observed in sections prepared in this manner (Figure 3(A) & Figure 3(D)).…”

“…Molecular biological approaches have been used in previous studies to assess the mechanism of formation of compression wood [10]- [15]. We demonstrated that: 1) expression of a laccase (CoLac1) in Chamaecyparis obtusa (a coniferous species) is very low in differentiating normal wood but very high in differentiating compression wood; 2) laccase activity is higher in the S2L region than in other parts of the S2 layer; 3) Colac1 is expressed at the onset of secondary wall thickening and is localized to the S2L region [16] [17]. Based on these results, we conclude that a specific laccase (CoLac1) is localized in the S2L region, which results in a high lignin concentration in the S2L region in C. obtusa.…”

Immunolocalization of a Normal Wood Specific Pectin Methylesterase (CoPME) and Quantification of PME Gene Expression in Differentiating Xylem of <i>Chamaecyparis obtusa</i>

“…[33]. Transverse thin (0.5 µm) sections for immunofluorescence light microscopy were cut using a rotary microtome (HM 350; MICROM, Walldorf, Germany) with a diamond knife and then mounted on MAS-GP typeA slides (Matsunami, Osaka, Japan).…”