Summary The woody stems of coniferous gymnosperms produce specialised compression wood to adjust the stem growth orientation in response to gravitropic stimulation. During this process, tracheids develop a compression‐wood‐specific S2L cell wall layer with lignins highly enriched with p‐hydroxyphenyl (H)‐type units derived from H‐type monolignol, whereas lignins produced in the cell walls of normal wood tracheids are exclusively composed of guaiacyl (G)‐type units from G‐type monolignol with a trace amount of H‐type units. We show that laccases, a class of lignin polymerisation enzymes, play a crucial role in the spatially organised polymerisation of H‐type and G‐type monolignols during compression wood formation in Japanese cypress (Chamaecyparis obtusa). We performed a series of chemical‐probe‐aided imaging analysis on C. obtusa compression wood cell walls, together with gene expression, protein localisation and enzymatic assays of C. obtusa laccases. Our data indicated that CoLac1 and CoLac3 with differential oxidation activities towards H‐type and G‐type monolignols were precisely localised to distinct cell wall layers in which H‐type and G‐type lignin units were preferentially produced during the development of compression wood tracheids. We propose that, not only the spatial localisation of laccases, but also their biochemical characteristics dictate the spatial patterning of lignin polymerisation in gymnosperm compression wood.

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.

Special xylem tissue called "compression wood" is formed on the lower side of inclined stems when gymnosperms grow on a slope. We investigated the molecular mechanism of compression wood formation. Transcriptome analysis by next-generation sequencing (NGS) was applied to the xylem of Chamaecyparis obtusa to develop a catalog of general gene expression in differentiating xylem during compression and normal wood formation. The sequencing output generated 234,924,605 reads and 40,602 contigs (mean size = 529 bp). Based on a sequence similarity search with known proteins, 54.2% (22,005) of the contigs showed homology with sequences in the databases. Of these annotated contigs, 19,293 contigs were assigned to Gene Ontology categories. Differential gene expression between the compression and normal wood libraries was analyzed by mapping the reads from each library to the assembled contigs. In total, 2875 contigs were identified as differentially expressed, including 1207 that were up-regulated and 1668 that were down-regulated in compression wood. We selected 30 genes and compared the transcript abundance between compression and normal wood by quantitative polymerase chain reaction analysis to validate the NGS results. We found that 27 of the 30 genes showed the same expression patterns as the original NGS results.

Woody plants develop a specialized secondary xylem known as reaction wood to enable formation of an ideal shape. Reaction wood in coniferous species is known as compression wood, and that of woody angiosperms as tension wood. However, the genus Buxus which is classified as an angiosperm, forms compression-wood-like reaction wood. We investigated the mechanism of lignification in coniferous compression wood and Buxus reaction wood: 1) Several lignin synthesis genes were upregulated in differentiating reaction wood of Buxus microphylla; 2) B. microphylla possesses a specific laccase gene that is expressed specifically in differentiating reaction wood (BmLac4); 3) laccase activity localization was closely related to lignification of reaction wood, and laccase activity was high in the secondary wall middle layer; 4) in reaction wood cell walls, galactan was present in the outer portion of the secondary wall middle layer, and the level of xylan was reduced. These findings suggest that lignification in B. microphylla reaction wood is identical to that of coniferous compression wood. These may represent general mechanisms of increasing lignin content in various reaction woods.

Lignin is a major component of plant cell walls and is synthesised through oxidative polymerisation of monolignols. The transcription level of laccase, an enzyme implicated in monolignol polymerisation, is higher in the tissue forming compression wood than in normal wood. Compression wood, which is a special xylem tissue that develops to reorient inclined stems, also has a higher lignin content than normal wood. In the present study, Chamaecyparis obtusa Endl. saplings were grown on an incline and the following variables were tracked for 10 weeks: posture recovery of the saplings; development of xylem tissue on the lower side of inclined stems; and the transcription level of laccase. The posture of saplings approached vertical after 8 weeks, the development of compression wood reached a peak around 6 weeks and laccase transcription was the highest after 4 weeks. These results suggest a sequence of righting mechanisms. Inclination stimulates an increase in the abundance of laccase transcript and this increase encourages the formation of compression wood. The accumulation of compression wood then causes the stem to bend upward.