Laccases are encoded by multigene families in plants. Previously, we reported the cloning and characterization of five divergent laccase genes from poplar (Populus trichocarpa) xylem. To investigate the role of individual laccase genes in plant development, and more particularly in lignification, three independent populations of antisense poplar plants, lac3AS, lac90AS, and lac110AS with significantly reduced levels of laccase expression were generated. A repression of laccase gene expression had no effect on overall growth and development. Moreover, neither lignin content nor composition was significantly altered as a result of laccase suppression. However, one of the transgenic populations, lac3AS, exhibited a 2-to 3-fold increase in total soluble phenolic content. As indicated by toluidine blue staining, these phenolics preferentially accumulate in xylem ray parenchyma cells. In addition, light and electron microscopic observations of lac3AS stems indicated that lac3 gene suppression led to a dramatic alteration of xylem fiber cell walls. Individual fiber cells were severely deformed, exhibiting modifications in fluorescence emission at the primary wall/middle lamella region and frequent sites of cell wall detachment. Although a direct correlation between laccase gene expression and lignification could not be assigned, we show that the gene product of lac3 is essential for normal cell wall structure and integrity in xylem fibers. lac3AS plants provide a unique opportunity to explore laccase function in plants.

Transgenic maize (Zea mays) plants were generated with a construct harboring a maize caffeic acid O-methyltransferase (COMT) cDNA in the antisense (AS) orientation under the control of the maize Adh1 (alcohol dehydrogenase) promoter. Adh1-driven ␤-glucuronidase expression was localized in vascular tissues and lignifying sclerenchyma, indicating its suitability in transgenic experiments aimed at modifying lignin content and composition. One line of AS plants, COMT-AS, displayed a significant reduction in COMT activity (15%-30% residual activity) and barely detectable amounts of COMT protein as determined by western-blot analysis. In this line, transgenes were shown to be stably integrated in the genome and transmitted to the progeny. Biochemical analysis of COMT-AS showed: (a) a strong decrease in Klason lignin content at the flowering stage, (b) a decrease in syringyl units, (c) a lower p-coumaric acid content, and (d) the occurrence of unusual 5-OH guaiacyl units. These results are reminiscent of some characteristics already observed for the maize bm3 (brownmidrib3) mutant, as well as for COMT down-regulated dicots. However, as compared with bm3, COMT down-regulation in the COMT-AS line is less severe in that it is restricted to sclerenchyma cells. To our knowledge, this is the first time that an AS strategy has been applied to modify lignin biosynthesis in a grass species.Lignins are complex phenolic polymers present in all vascular plants. They provide rigidity to conducting xylem elements and fiber cells. Lignins are composed of C 6 C 3 units, principally p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units, and are present in various proportions according to botanical, physiological, and cytological criteria (Lewis and Yamamoto, 1990). Throughout the plant kingdom, grass lignins appear to be particularly specialized because they contain not only H, G, and S units, but also additional p-hydroxycinnamic units such as p-coumaric and ferulic acids (Higuchi et al., 1967). Ferulic acid may be ester linked to wall polysaccharides and/or ether linked to G units, thereby forming bridges between lignins and polysaccharides (Jacquet et al., 1995), whereas p-coumaric acid is primarily ester linked to S lignin units in lignified walls (Ralph et al., 1994; Grabber et al., 1996).Lignification in dicotyledons has been extensively studied and most of the known lignin biosynthetic genes have been employed in genetic engineering experiments (for review, see Grima-Pettenati and Goffner, 1999). There are almost no molecular data on lignification in grasses (Collazo et al., 1992;McAlister et al., 1998;Pichon et al., 1998;Selman-Housein et al., 1999;Spangenberg et al., 2001). Although lignification in grass species is likely to share a high degree of similarity to other angiosperms, the aforementioned structural specificity of grass cell walls may also involve a certain degree of grass-specific regulatory mechanisms. An in-depth knowledge of lignification in Graminaeae is of utmost importance because lignins are one of the ...