Transgenic poplars (Populus tremula ×Populus alba) were obtained by introduction of a sense homologous transgene encoding caffeic acidO-methyltransferase (COMT) under the control either of the cauliflower mosaic virus double 35S promoter or of the eucalyptus cinnamyl alcohol dehydrogenase promoter. Although these constructs conferred a moderate overexpression of COMT in some lines, a transgenic line with the double 35S promoter was found where COMT activity in woody tissues was close to zero due to a gene-silencing phenomenon. For the first time in COMT down-regulated trees, this alteration substantially reduced lignin level in 6-month-old trees (17% decrease). Lignin structure was found to be strongly altered, with a two times higher content in condensed bonds, an almost complete lack of syringyl units, and the incorporation of 5-hydroxyguaiacyl units to the most remarkable extent reported so far. Consistent with the higher cellulose content and with the higher condensation degree of the lignin, the impact of the transformation on the kraft-pulping performances of the poplar trees positively affected the pulp yield (10% relative increase), but made lignins less amenable to industrial degradations.

). ² These authors contributed equally to this work. SummaryUsing tobacco transgenic lines altered in the monolignol biosynthetic pathway and which differ in their lignin pro®les we have evaluated lignin deposition at the cellular and subcellular levels using several microanalytical techniques. Surprisingly, whereas a Cinnamoyl CoA reductase (CCR) down-regulated line with a strong decrease in lignin content exhibited an overall reduction in lignin deposition in the walls of the different xylem cell types, this reduction was selectively targeted to the ®bers in a double transformant (down-regulated for both CCR and Cinnamyl alcohol dehydrogenase (CAD)) displaying a similar degree of global lignin content decrease. Fiber and vessel secondary walls of the transgenic tobacco line homozygous for the ccr antisense gene (CCR.H) down-regulated plants were dramatically destructured, particularly in the S2 sublayer, whereas the deposition of lignins in the S1 sublayer was not signi®cantly modi®ed. In contrast, cell wall organization was slightly altered in xylem cells of the double transformant. The relative distribution of non-condensed and condensed units in lignin, evaluated microscopically with speci®c antibodies, was differentially affected in the transgenics studied and, in a general way, a drop in non-condensed lignin units (b± 0±4 interunit linkages) was associated with a loss of cohesion and extensive disorganization of the secondary wall. These results demonstrate that ligni®cation is tightly and independently regulated in individual cell types and cell wall sublayers. They also show that down-regulation of speci®c genes may induce targeted changes in lignin structure and in spatial deposition patterns of the polymer.

No abstract

Lignin biosynthesis is an essential physiological activity of vascular plants if they are to survive under various environmental stresses on land. The biosynthesis of lignin proceeds in the cell wall by polymerization of precursors; the initial step of lignin polymerization is the transportation of lignin monomers from the cytosol to the cell wall, which is critical for lignin formation. There has been much debate on the transported form of the lignin precursor, either as free monolignols or their glucosides. In this study, we performed biochemical analyses to characterize the membrane transport mechanism of lignin precursors using angiosperms, hybrid poplar (Populus sieboldii × Populus grandidentata) and poplar (Populus sieboldii), as well gymnosperms, Japanese cypress (Chamaecyparis obtusa) and pine (Pinus densiflora). Membrane vesicles prepared from differentiating xylem tissues showed clear ATP-dependent transport activity of coniferin, whereas less than 4% of the coniferin transport activity was seen for coniferyl alcohol. Bafilomycin A1 and proton gradient erasers markedly inhibited coniferin transport in hybrid poplar membrane vesicles; in contrast, vanadate had no effect. Cis-inhibition experiments suggested that this transport activity was specific for coniferin. Membrane fractionation of hybrid poplar microsomes demonstrated that transport activity was localized to the tonoplast- and endomembrane-rich fraction. Differentiating xylem of Japanese cypress exhibited almost identical transport properties, suggesting the involvement of a common endomembrane-associated proton/coniferin antiport mechanism in the lignifying tissues of woody plants, both angiosperms and gymnosperms.

Lignin distribution in the tracheid cell wall of mild compression wood in Pinus radiata D. Don was examined by interference microscopy, confocal fluorescence microscopy, and ultraviolet (UV) microscopy. Two anatomically different samples of mild compression wood were compared with a sample of normal wood using quantitative interference microscopy and microdensitometry combined with confocal fluorescence microscopy to estimate the quantitative or semiquantitative lignin distribution in the S2 and S2L regions of the secondary cell wall and of the cell corner middle lamella (CCML). One of these samples was briefly examined by UV microscopy for comparison. Quantitative interference microscopy provided information on lignin concentration in different regions of the cell wall with values of 26, 46, and 57%, respectively, for the S2, S2L, and CCML regions of sample 1 and 20, 29, and 46%, respectively, for the same regions of sample 2. Microdensitometry of confocal fluorescence images provided semiquantitative information on the relative lignin distribution based on lignin autofluorescence. Comparison between the two compression wood samples using autofluorescence gave results that were in partial agreement with interference microscopy with respect to the relative lignification levels in the S2, S2L, and CCML regions. Some improvement was achieved by using calibration values for hemicellulose rather than holocellulose for interference data in the S2L region. Results for UV microscopy performed on sample 1 indicated that the lignification of the CCML region was comparable with that of the S2L region in this sample but with some variation among cells. All three techniques indicated significant variation in lignification levels of the S2L and CCML regions among adjacent cells and a significant reduction in the lignification of the CCML region compared to normal wood.Key words: lignin distribution, interference microscopy; confocal fluorescence microscopy, UV microscopy, mild compression wood, Pinus radiata D. Don.