Trideuterated vanillin and hexadeuterated syringaldehyde were prepared respectively by monomethylation of 3,4-dihydroxybenzaldehyde [27] and copper assisted methoxy substitution of 3,5-dibromo-4-hydroxy-benzaldehyde [28].

Trideuterated coniferin and hexadeuterated syringin were prepared as previously reported [26].

Trideuterated coniferin spectrometric data: ¹H NMR (CD₃OD): δ=7.04 (d, 1 H, H_arom2, J=2Hz), 7.01 (d, 1, H_arom5, J=8.2Hz), 6.87 (dd, 1 H, H_arom6, J=8.2Hz; 2 Hz), 6.54 (d, 1 H, ArCHCH, J=15.9Hz), 6.25 (dt, 1 H, ArCHCH, J=15.9Hz; 5.6 Hz), 4.86 (d, 1 H, H1, J=7.0Hz), 4.08 (d, 2 H, ArCHCHCH₂, J=5.6Hz), 3.75 (m, 1 H, H6b), 3.58 (m, 1 H, H6a), 3.27–3.43 (m, 4 H, H2,3,4,5). MS (EI, 70 eV) m/z (%): 183 (100), 140 (79), 131 (23), 127 (78), 119 (30), 109 (30), 107 (26), 105 (24), 103 (34), 92 (26), 91 (75), 89 (27), 85 (22), 81 (32), 79 (36), 78 (32), 77 (66), 73 (51), 71 (28), 69 (31), 63 (23), 60 (47), 57 (53), 56 (26), 55 (87). C₁₆H₁₉D₃O₈.(H₂O)₂: C: 50.24 (calcd 50.39), H: 6.68 (calcd 6.87).

Hexadeuterated syringin spectrometric data: ¹H NMR (DMSO, d6): δ=6.71 (s, 2 H, H_arom2,6), 6.45 (d, 1 H, ArCHCH, J=15.9Hz), 6.30 (dt, 1 H, ArCHCH, J=15.9Hz; 4.9 Hz), 4.90 (d, 1 H, H1, J=6.7Hz), 4.10 (d, 2 H, ArCHCHCH₂, J=4.9Hz), 3.58 (m, 1 H, H6b), 3.40 (m, 1 H, H6a), 3.00–3.25 (m, 4 H, H2,3,4,5). MS (EI) m/z(%): 217 (33), 216 (100), 188 (22), 187 (10), 173 (27), 160 (17), 152 (12), 73 (11), 60 (11); HRMS: calcd: 379.1875, found 379.1900.

2.2.1 Plant labelling and culture

2.2.2 Sampling procedure

Root or stem samples were washed with methanol, dried and then subjected to thioacidolysis as previously described [12]. The lignin-derived thioacidolysis monomers were trimethylsilylated and injected for GC-MS analysis on a Varian Saturn IV (Varian, Palo Alto, USA) ion trap mass spectrometer using the conditions previously described [9].

Hand sections of 16 week-old plantlets, displaying about a dozen of internodes and fully developed leaves, were made using a razor blade. To detect native lignins from their cinnamaldehyde end-groups, these sections were treated with phloroglucinol, 2% (w/v) in ethanol, mixed 2:1 with concentrated hydrochloric, which revealed lignified areas from their purple colour [30]. To detect syringyl units in lignins, the Mäule staining was carried out by immersing the sections in a 1% (w/v) water solution of potassium permanganate for 5 min. The sections were then thoroughly rinsed in distilled water and treated with 3% hydrochloric acid until the dark colour faded, after which concentrated ammonium hydroxide was added to the sections to develop a magenta colour in syringyl lignified tissues [30]. These sections were done at the top (penultimate internode from the top), at the middle (fifth internode from the top) and at the base (tenth internode from the top) of the stem plantlets.

To visualise the lignified tissues in 16 week-old plantlet stems, cross sections of top, middle and bottom internodes were treated with the Wiesner (phloroglucinol–HCl) and the Maüle reagents. Phloroglucinol staining has long been utilized for localizing lignins in plant tissue sections [30]. It stains lignins a purple coloration deriving from reactions of hydroxycinnamyl aldehyde end-groups that are systematically present in native lignins, albeit in low amounts. The Maüle reagent specifically stains bright red the S lignin units. In agreement with literature data [31], the stems regenerated from the bud displayed a fully developed vascular cambium at the basal and middle parts of the 16 week-old plantlets whereas the top internode only displayed primary vascular bundles.

When the middle stem cross-sections were subjected to the phloroglucinol–HCl staining, the xylem of the control plants stained more importantly than that of the ASOMT2B ones (Fig. 2). In addition, in the top internodes provided only with primary vascular bundles, the WT sample stained red purple, which is diagnostic for lignified cell walls. In contrast, the vascular bundles of the ASOMT2B top sections did not stain, which suggests that they are not lignified or not lignified enough for a positive Wiesner coloration. Not unexpectedly, lignin in the secondary xylem of the basal and middle stem cross-sections of the WT line exhibited the positive magenta coloration diagnostic of S units while the analogous ASOMT2B sections showed a reduced staining, indicating a reduction in S lignin units (Fig. 2), which is consistent with the genetically-reduced COMT activity [17]. Taken together, these observations suggest that the COMT reduced activity not only decrease the frequency of S lignin units, but also induces some delay in lignification.

The deuterated coniferin and syringin used for the set of experiments described herein were prepared as previously described in details [26]. The employed method is based on the coupling of a glucosidation and a Wittig reaction, leading to α,β-unsaturated aldehydes, then easily reduced to the alcohols [32].

The coniferin and syringin deuterated at their methoxyl groups carried three or six deuterons, respectively. These deuterium atoms on methoxyl groups are not prone to exchange during lignin biosynthesis. Their incorporation into G or S lignin units was monitored by GC-MS examination of the base peaks of the mass spectra of G and S thioacidolysis monomers. In the unlabelled G and S monomers recovered from WT and ASOMT2B samples not fed with deuterated precursors, the base peaks were respectively observed at m/z 269 and 299, which corresponds to the benzylic ions G-CHSEt⁺ and S-CHSEt⁺ (with G and S = trimethylsilylated guaiacyl and syringyl rings). The natural isotopic patterns for these unlabeled G and S ions were repeatedly found to give (M+3) to M ratio lower than 2% while the (M+6) to M ratio of the S compound was found close to zero. Accordingly, the incorporation of tri- or hexa-deuterated G and S rings into the lignins of deuterium fed plantlets could be traced out from abnormally high M+3 or M+6 signals.

When added to the solid culture medium, deuterated coniferin was massively used for the biosynthesis of root lignins, as revealed by the recovery of high deuterium level in thioacidolysis monomers released from the roots of WT and ASOMT2B plantlets (Table 1). In contrast, stem lignins were found to be deuterium-labelled to a much lower extent. This result establishes that coniferin diffuses into the gelose medium, is absorbed by the roots and then migrates essentially to the lignifying root cell walls where its aglycone is used for lignin biosynthesis. The administration way proposed in this study seems therefore more adapted to the study of root lignification. The deuterium labelling of root lignins could be evidenced not only in G units but also, more markedly, in S units. Deuterium incorporation into root lignins increases between the fourth and the tenth week after the beginning of the plantlet culture. In the following weeks, this deuterium labelling of root lignins was found to decrease. In other words, the effective use of the deuterated coniferin added to the medium lasted at least over a 10-week-long period. COMT deficiency in the ASOMT2B line did not seem to impede the use of deuterated coniferin. The only visible difference between the WT and transgenic samples was the higher deuterium level in the roots of the 4 and 16 week-old plantlets.

The syringin incorporation was performed in parallel to another coniferin feeding, using a different administration procedure (addition of the precursors at 0, 1 and 4 weeks after the beginning of the in vitro culture). No major difference, but a slightly higher deuterium concentration was observed when trideuterated coniferin was fed in three portions, delayed by few weeks. The highest deuterium incorporation in stem lignins was obtained with three administrations of [3,5-OCD₃]-syringin, with which hexadeuterated S units reach 23% (Table 2). Different explanations can account for this result. Syringin may enter or migrate more easily than coniferin. In addition, since the deuterium is found only in S lignin units (Table 2), it means that is not divided between G and S units, contrarily to the deuterium of coniferin.

Taken together, the data of Tables 1 and 2 reveal that the deuterium of syringin was exclusively directed to syringyl lignin units, whereas the deuterium of coniferin could be evidenced not only in G lignin units, but also and more markedly in S lignin units. In agreement with recent studies [22–24], this result further establishes that the hydroxylation/methylation steps leading to the formation of the S aromatic ring may occur at the alcohol stage and not only at the aldehyde stage [21].

The total yield and relative ratio of the G and S thioacidolysis monomers reflects the total lignin amount and the relative frequency of the G and S lignin units (Table 3), without interference from non-lignin components. For all plantlet roots, the (S + G) yield stabilized around 100 μmol per dry weight gram. As the recovery yield of thioacidolysis monomers released from 1 g of native poplar lignins varies between 1000 and 2000 μmol per dry weight gram [2,12], we may roughly estimate the root lignin content of the poplar plantlets varying between 5 and 10% (weight percentage). This approximation reveals that plantlet roots are substantially lignified. In addition, it should be kept in mind that the S and G thioacidolysis monomers may originate not only from lignin, but also from the aromatic domain of suberin, as both lignin and suberin have been reported to be important components of the root apoplastic barriers [33]. A fairly stable S/G thioacidolysis ratio, close to 0.4, was observed for all the root plantlet samples, whatever the line. That WT and ASOMT2B root samples yield similar (S + G) and S/G thioacidolysis values indicates that COMT deficiency has no impact on root lignification of in vitro grown poplar plantlets. This observation could be related to the lower fact that COMT is less repressed in the root than in the stem of the ASOMT2B line (Jouanin, unpublished results). The most important result revealed by the kinetic monitoring of lignification in the unlabelled plantlets and in the plantlets fed with deuterated coniferin or syringin is the following one. While the effective and massive incorporation of deuterium in G and S root lignin units could be assessed, this incorporation had no obvious impact on the lignin content and structure that were found to be very similar in the unlabelled and in the labelled samples. In other words, plantlets used exogenous material to synthesize root lignins, without any modification of lignin content or S/G ratio. When deuterated coniferin was fed to the plantlet, the S units of root lignins were substantially more deuterated than the G units (Table 1). The conversion of G rings into S rings at the stage of p-OH cinnamyl alcohols therefore allowed the plantlet to redirect the G-type lignin precursor towards the synthesis of S lignin units so that the S/G ratio is maintained at a genetically-programmed value in the unlabelled or labelled samples. The lignin content and structure seem therefore to be under the control of a tight regulation mechanism.

In contrast to root lignification and in agreement with a wealth of literature data [17,18,34], stem lignification was found to be affected by COMT deficiency. The total thioacidolysis yields were systematically found to be lower in the ASOMT2B stems than in the WT analogues (Table 3). The most likely explanation for these lower thioacidolysis yields and in agreement with the aforementioned reduced Wiesner staining is that the ASOMT2B stems are less lignified than their WT counterparts. In addition, and in agreement with the reduced Maüle staining, the ASOMT2B stem lignins displayed a reduced frequency of S lignin units when compared to the control. This result confirms that the biosynthesis of S lignin units is affected by COMT deficiency at the very early stages of lignification in growth-chamber-cultivated plantlets regenerated from microcuttings. The 5-OH guaiacyl (5-OH G) units which are the diagnostic signature of COMT deficiency were found in trace amounts before the 12th week of in vitro culture. It was only in the 16 week-old plantlets that the frequency of these units slightly increased relative to the control value (data not shown).