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Keywords:

  • Pinus radiata;
  • caffeoyl-CoA 3-O-methyltransferase;
  • tracheary elements;
  • lignin;
  • caffeyl alcohol;
  • benzodioxane

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusion
  7. Experimental procedures
  8. Acknowledgements
  9. References
  10. Supporting Information

A cDNA clone encoding the lignin-related enzyme caffeoyl CoA 3-O-methyltransferase (CCoAOMT) was isolated from a Pinus radiata cDNA library derived from differentiating xylem. Suppression of PrCCoAOMT expression in P. radiata tracheary element cultures affected lignin content and composition, resulting in a lignin polymer containing p-hydroxyphenyl (H), catechyl (C) and guaiacyl (G) units. Acetyl bromide-soluble lignin assays revealed reductions in lignin content of up to 20% in PrCCoAOMT-deficient transgenic lines. Pyrolysis-GC/MS and 2D-NMR studies demonstrated that these reductions were due to depletion of G-type lignin. Correspondingly, the proportion of H-type lignin in PrCCoAOMT-deficient transgenic lines increased, resulting in up to a 10-fold increase in the H/G ratio relative to untransformed controls. 2D-NMR spectra revealed that PrCCoAOMT suppression resulted in formation of benzodioxanes in the lignin polymer. This suggested that phenylpropanoids with an ortho-diphenyl structure such as caffeyl alcohol are involved in lignin polymerization. To test this hypothesis, synthetic lignins containing methyl caffeate or caffeyl alcohol were generated and analyzed by 2D-NMR. Comparison of the 2D-NMR spectra from PrCCoAOMT-RNAi lines and synthetic lignins identified caffeyl alcohol as the new lignin constituent in PrCCoAOMT-deficient lines. The incorporation of caffeyl alcohol into lignin created a polymer containing catechyl units, a lignin type that has not been previously identified in recombinant lignin studies. This finding is consistent with the theory that lignin polymerization is based on a radical coupling process that is determined solely by chemical processes.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusion
  7. Experimental procedures
  8. Acknowledgements
  9. References
  10. Supporting Information

Lignin is a heterogeneous cell-wall polymer created though oxidative coupling of p-hydroxycinnamyl alcohols (monolignols) or related compounds (Ralph et al., 2004). The polymer is particularly abundant in wood-forming cells such as tracheids, wood fibers and vessel elements that undergo secondary cell-wall thickening (Harris, 2005). It is covalently linked to non-cellulosic cell-wall polysaccharides and plays vital roles in vascular plants as it reinforces plant cell walls, facilitates water transport, provides compressive strength to conducting tissues, and acts as a mechanical barrier to pathogens (Boudet, 2007).

Genetic and biochemical studies over the last 15 years have resulted in substantial revisions and refinements of the monolignol biosynthetic pathway (Umezawa, 2010). This research has also provided new insights into the consequences of lignin manipulation on its content and composition (Boerjan et al., 2003; Halpin, 2004; Chiang, 2006; Higuchi, 2006; Boudet, 2007; Rastogi and Dwivedi, 2008; Vanholme et al., 2008; Umezawa, 2010). More recently, the interfering role that lignin plays in biomass utilization, including chemical pulping, forage digestibility and conversion of plant biomass to biofuels, has further stimulated lignin research (Halpin, 2004; Ralph et al., 2004; Chapple et al., 2007; Chen and Dixon, 2007; Vanholme et al., 2008, 2010; Hisano et al., 2009; Simmons et al., 2010).

Perturbations of the monolignol pathway can lead to structural changes in the lignin polymer that are the consequence of incorporation of chemical intermediates in the monolignol biosynthetic pathway into the lignin polymer (Ralph et al., 2001, 2004, 2008; Boerjan et al., 2003; Lapierre et al., 2004; Morreel et al., 2004). Such data support the hypothesis that lignin polymerization is stochastic, but results from a radical coupling process that is under chemical control (Ralph et al., 2004, 2008). The fact that monolignols are cross-coupled onto the growing polymer in a chemically controlled fashion may not only facilitate manipulation of lignin composition but also enable the ‘design’ of a lignin polymer for easier biomass processing (Grabber et al., 2008, 2010; Ralph, 2010; Simmons et al., 2010).

Coniferous gymnosperms such as pines differ in many anatomical features from most angiosperms, and this includes the cellular composition of wood. The physiological and structural roles of vessel elements and wood fibers in angiosperms are performed by tracheids in coniferous gymnosperms. The biochemical composition of tracheid cell walls differs substantially from those of vessel elements and wood fibers, particularly with regard to non-cellulosic polysaccharides and lignin (Harris, 2005). Lignin in conifers is primarily derived from the monolignols p-coumaryl alcohol (H-type units) and coniferyl alcohol (G-type units), and lacks the sinapyl alcohol-derived subunits (S-type) that are commonly found in angiosperms (Harris, 2005). Consistent with the differences in wood anatomy between angiosperm and gymnosperm species, functional genomics studies targeting monolignol and lignin biosynthesis in gymnosperms have produced phenotypes that have not been previously observed in angiosperms (Möller et al., 2005; Wagner et al., 2007, 2009; Wadenbäck et al., 2008).

Callus cultures capable of producing tracheary elements (TEs) are excellent experimental platforms to investigate xylogenesis-related processes, including secondary cell-wall formation and lignification (Kärkönen and Koutaniemi, 2010). The differentiation of TEs follows the same developmental pattern observed in tracheids, and includes cell expansion, secondary cell-wall deposition and programmed cell death (Turner et al., 2007). TE cultures are versatile, because they are independent of seasonal effects and are amenable to metabolic manipulations by feeding metabolites or inhibitors (Kärkönen and Koutaniemi, 2010). Most suitable for functional genomics studies are TE cultures that can be transformed and subsequently induced to differentiate tracheary elements (Möller et al., 2003; Oda et al., 2005). We have developed such a tissue culture system for Pinus radiata (Möller et al., 2003). We previously demonstrated that the biochemical cell-wall composition of developed TEs in P. radiata is similar to that of wood tracheids (Möller et al., 2006). We have exploited this fact to perform functional genomic studies of the lignin biosynthetic pathway (Möller et al., 2005; Wagner et al., 2007).

One of the key enzymes involved in the biosynthesis of monolignols is caffeoyl-CoA 3-O-methyltransferase (CCoAOMT). In angiosperms, this enzyme is required for the biosynthesis of G- and S-type lignins (Meyermans et al., 2000; Zhong et al., 2000; Marita et al., 2003; Do et al., 2007). Its preferred substrate in all angiosperm species analyzed so far is caffeoyl-CoA, which is converted into feruloyl-CoA (Figure 1). CCoAOMT suppression in Nicotiana tabacum, Arabidopsis thaliana, Medicago sativa and Populus tremula × alba caused lignin reductions of 20–45% (Meyermans et al., 2000; Zhong et al., 2000; Marita et al., 2003; Do et al., 2007). These studies all reported that suppression of CCoAOMT compromised formation of both G- and S-type lignin, consistent with caffeoyl-CoA being a precursor of G- and S-type lignin in angiosperms.

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Figure 1.  Enzymatic reaction supported by caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) in monolignol biosynthesis. SAM, S-adenosyl methionine; SAH, S-adenosyl homocysteine.

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In Pinus taeda, CCoAOMT is preferentially expressed in differentiating xylem (Li et al., 1999). As in angiosperms, in vitro studies suggested that the preferred substrate of the recombinant enzyme is caffeoyl-CoA (Li et al., 1999). In silico analysis indicated that CCoAOMT could potentially be encoded by a small gene family in conifers (Friedmann et al., 2007; Koutaniemi et al., 2007). However, experiments to test the biological function of these genes and their involvement in lignin biosynthesis have not been undertaken to date.

Here we describe the isolation of a putative P. radiata CCoAOMT clone from a cDNA library derived from differentiating xylem, and the effects of its suppression on TE-forming P. radiata callus cultures. Our results demonstrate that the isolated CCoAOMT clone (PrCCoAOMT) is involved in biosynthesis of G-type lignin in coniferous gymnosperms such as P. radiata, and that suppression of this gene results in a lignin polymer consisting of p-hydroxyphenyl (H), catechyl (C) and guaiacyl (G) subunits.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusion
  7. Experimental procedures
  8. Acknowledgements
  9. References
  10. Supporting Information

PrCCoAOMT isolation, generation and screening of transgenic lines

An 805 bp fragment of a putative P. radiata CCoAOMT clone (PrCCoAOMT; GenBank accession number HQ444753) containing the full-length 780 bp open reading frame was isolated from a cDNA library derived from differentiating xylem using the PCR-based approach described in Experimental procedures. The deduced amino acid sequence of the isolated clone was approximately 98% identical to its presumed Pinus taeda ortholog (GenBank accession number AF036095; Li et al., 1999), and approximately 93% identical to putative CCoAOMT clones from other conifers such as Picea abies (GenBank accession number CAK18782.1), Picea glauca (GenBank accession number GQ03207_P07) and Picea sitchensis (GenBank accession number WS02736_J21) (Figure S1). Quantitative RT-PCR experiments revealed that the expression of PrCCoAOMT increased approximately 10-fold during TE development in concert with other lignin-related genes (Wagner et al., 2007).

Biolistic co-transformation of P. radiata TE cultures with plasmids pLTK4 and pAW16 (Figure 2) resulted in the generation of 36 transgenic lines. Genomic integration of the PrCCoAOMT RNAi construct was verified by PCR (data not shown), and approximately 80% of all transgenic lines tested contained both the 867 and 1870 bp DNA fragments shown in Figure 2. Twenty-one of these transgenic lines were induced to differentiate into TEs to assess potential phenotypes based on PrCCoAOMT suppression. Differentiated pine callus cultures contain a mixture of non-differentiated cells, developing TEs and fully differentiated TEs (Figure 3). Chemical fingerprints from such cell mixtures were generated by pyrolysis-GC/MS as an initial screen for phenotypic changes in transgenic material. A comparative analysis of the pyrolysis-GC/MS spectra (pyrograms) from the 21 PrCCoAOMT RNAi lines with those from 16 control lines revealed phenotypic differences in cell-wall composition in approximately one-third of the transgenic lines (data not shown). The most obvious phenotypic trend in PrCCoAOMT RNAi lines was a two- to threefold higher H/G ratio. Three transgenic lines with this phenotypic trend, pLTK4-22, pLTK4-26 and pLTK4-35, were chosen for more detailed studies. Quantitative RT-PCR experiments with these transgenic lines revealed an 84–90% reduction in the PrCCoAOMT steady-state RNA levels compared to wild-type controls (Table S1).

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Figure 2.  Schematic diagrams of the constructs used in this study to transform Pradiata callus cultures. Plasmid pLTK4 contains an inverted repeat of the PrCCoAOMT coding region separated by the Zmays UBI1 intron and under the control of the Zmays UBI1 promoter; plasmid pAW16 contains the NPTII resistance gene under the control of the Zea mays UBI1 promoter and the GUS reporter gene under the control of a double CaMV 35S promoter. The positions of the 867 and 1870 bp PCR fragments used to confirm genomic integration of the PrCCoAOMT RNAi construct are indicated.

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image

Figure 3.  Confocal fluorescence image of Pradiata TE cultures stained with Congo red and acriflavin. Non-differentiated cells and non-lignified TEs with secondary cell-wall thickening appear red. Lignin deposition in tracheary elements appears green. Scale bar = 25 μm.

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Quantitative changes in the lignin of PrCCoAOMT-deficient transgenic lines

Differentiated TEs produced in pLTK4-22, pLTK4-26 and pLTK4-35 and a wild-type control were further separated from non-differentiated material for the purpose of quantitative chemical analyses of cell-wall material as described previously (Wagner et al., 2007). This enrichment procedure routinely results in TE fractions that contain at least 90% differentiated TEs (data not shown), which is comparable to the proportion of tracheids in pine wood. Quantitative acetyl bromide-soluble lignin (ABSL) analysis with purified TE fractions derived from pLTK4-22, pLTK4-26 and pLTK4-35 revealed only moderate changes in lignin content compared to the wild-type control. A reduction in lignin content of approximately 20% was observed in line pLTK4-22. The other two transgenic lines exhibited reductions in lignin content of between 5 and 16% compared to a wild-type control of the same genotype (Table 1).

Table 1.   ABSL lignin (% w/w) in TEs of a wild-type control and PrCCoAOMT-deficient lines pLTK4-22, pLTK4-26 and pLTK4-35
 Wild-typepLTK4-22pLTK4-26pLTK4-35
  1. *Values are means ± standard deviation of at least two independent measurements.

ABSL lignin26.5 ± 1.2*21.0 ± 0.122.3 ± 0.125.2 ± 0.3

Pyrolysis GC/MS experiments were performed with the same TE fractions that were previously used in ABSL assays. These experiments confirmed the changes in lignin content observed in ABSL assays and the trends in the H/G ratio detected in the pyrolysis screening experiment, particularly in line pLTK4-22, which displayed the strongest phenotype in ABSL lignin measurements (Table 1). Most notable was a decrease in pyrolysis products derived from G-type lignin, such as 4-ethyl-guaiacol, eugenol, 4-vinyl-guaiacol, cis- and trans-isoeugenol, vanillin, acetoguaiacone and guaiacylacetone (Figure 4) (Faix et al., 1990). The levels of pyrolysis products such as 4-methyl phenol, which are indicative of H-type lignin (Faix et al., 1990), were either slightly increased or similar to wild-type levels in transgenic lines (Figure 4).

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Figure 4.  Pyrogram of powdered, freeze-dried Pradiata TEs from wild-type control (a) and PrCCoAOMT-deficient lines pLTK4-22 (b) and pLTK4-26 (c). The following lignin-related pyrolysis products are labeled: 1, unknown; 2, guaiacol; 3, 4-methyl-guaiacol; 4, phenol; 5, 4-ethyl-guaiacol; 6, dimethyl-phenol; 7, 4-methyl-phenol; 8, eugenol; 9, 4-vinyl-guaiacol; 10, cis-isoeugenol; 11, trans-isoeugenol; 12, vanillin; 13, unknown; 14, acetoguaiacone; 15, guaiacylacetone; 16, catechol. Signals representing H-type lignin (4 and 7) are elevated or similar to wild-type levels, and signals for G-type lignin (3, 5, 8, 10, 11, 12, 14 and 15) are reduced in the transgenic lines.

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Compositional and structural changes in the lignin of PrCCoAOMT-deficient transgenic lines

Transgenic line pLTK4-22, which displayed the strongest phenotype in ABSL lignin and pyrolysis GC/MS experiments, did not generate a sufficient quantity of TEs for 2D-NMR studies. However, the PrCCoAOMT-deficient lines pLTK4-26 and pLTK4-35 could be used for 2D 13C–1H correlation (HSQC) NMR studies designed to investigate compositional and structural changes in lignin of purified TEs.

For 2D-NMR analysis, ‘enzyme lignins’ were generated, so-called because lignin was enriched in these preparations by removal of a substantial proportion of the cell-wall polysaccharides by digestion with crude polysaccharidases (Chang et al., 1975; Wagner et al., 2007). These lignin preparations were then dissolved in DMSO/N-methylimidazole (Lu and Ralph, 2003), acetylated, and dissolved in CDCl3 for structural analysis by NMR.

The lignin monomer composition was measured by volume integration of contours in HSQC spectra as described previously (Ralph et al., 2006; Wagner et al., 2009; Ralph and Landucci, 2010). The two transgenic lines displayed an approximately five- to 10-fold higher H/G ratio compared to the wild-type control (Table 2), a result consistent with the trends observed in the pyrolysis GC/MS experiments (Figure 4). The relative increase in H-type lignin in pLTK4-26 and pLTK4-35 was most easily visualized when the guaiacyl-2 C/H correlations were set to be approximately equivalent (Figure 5) (Kim and Ralph, 2010). Line pLTK4-26 displayed a stronger phenotype than line pLTK4-35 in these NMR experiments, consistent with the results of quantitative RT-PCR, ABSL and pyrolysis GC/MS analyses.

Table 2.   NMR-derived H/G and inter-unit linkage data of acetylated enzyme lignin in TEs of a wild-type control and PrCCoAOMT-deficient lines pLTK4-26 and pLTK4-35
Sample%H%GH/G%A%B%C%D%J%X1Σβ-O-4
  1. H, p-hydroxyphenyl; G, guaiacyl; A, β-aryl ether (β-O-4); B, phenylcoumaran (β-5); C, resinol (β–β); D, dibenzodioxocin (β-O-4/5-5); J, benzodioxane (β-O-4); X1, cinnamyl alcohol end-group (see Figure 6 for structures).

Control0.599.50.560.224.612.52.7010.262.9
pLTK4-265.294.85.358.422.311.91.85.610.463.6
pLTK4-352.397.72.459.424.512.51.71.99.663.0
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Figure 5.  Partial short-range 13C–1H (HSQC) spectra (aromatic regions) of acetylated enzyme lignin isolated from Pradiata TEs. (a) Wild-type control; (b) PrCCoAOMT-deficient line pLTK4-26; (c) PrCCoAOMT-deficient line pLTK4-35.

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Structural changes in lignin are best described by comparing inter-unit and end-unit profiles in HSQC spectra. Table 2 shows relative quantitative data derived from integrating the correlations from functional lignin units, as previously described (Wagner et al., 2009). The units measured are shown in Figure 6, which displays partial HSQC spectra (side-chain region only) of acetylated enzyme lignin from pLTK4-26 and pLTK4-35 and a wild-type control. β-Aryl ether (A), phenylcoumaran (B) and resinol (C) units, as well as cinnamyl alcohol end-groups (X1), were present at comparable levels in transgenic lines and the wild-type control. The amount of dibenzodioxocin (D) units appeared to be slightly reduced in transgenic lines compared to the wild-type control (Table 2). Most strikingly different was the presence of benzodioxanes (J) in both transgenic lines, which were absent in the wild-type control (Figure 6 and Table 2). Benzodioxane levels were higher in transgenic line pLTK4-26 than line pLTK4-35, confirming the phenotypic trends observed previously (Figure 4 and Table 1). Benzodioxanes in lignin have previously been identified in caffeic acid O-methyltransferase (COMT)-deficient angiosperm species as a consequence of incorporating 5-hydroxyconiferyl alcohol into the lignin polymer (Morreel et al., 2004). The formation of benzodioxanes requires monolignols with an ortho-diphenol structure such as that in 5-hydroxyconiferyl alcohol (Figure S2) (Marita et al., 2003; Morreel et al., 2004; Lu et al., 2010). Based on the position of CCoAOMT in the monolignol pathway, we hypothesized that benzodioxanes in pLTK4-26 and pLTK4-35 could have arisen from incorporating caffeyl alcohol or caffeate into the lignin polymer. To test this hypothesis, synthetic lignins (‘dehydrogenation polymers’; DHPs) from a mixture of caffeyl alcohol and coniferyl alcohol, as well as from methyl caffeate and coniferyl alcohol, were prepared. Comparison of the resulting expanded short-range 13C–1H (HSQC) spectra from pLTK4-26 and pLTK4-35 with those of the DHPs indicated that benzodioxanes in transgenic lines most likely arose from incorporation of caffeyl alcohol into the lignin polymer (Figure 7).

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Figure 6.  Partial short-range 13C–1H (HSQC) spectra (side-chain regions) of acetylated enzyme lignin isolated from isolated from P. radiata TEs. (a) Wild-type control; (b) PrCCoAOMT-deficient line pLTK4-26; (c) PrCCoAOMT-deficient line pLTK4-35; (d) differential spectrum [(b)–(a)]; (e) differential spectrum [(c)–(a)]. The differential spectra highlight new signals for benzodioxane units (J) in transgenic lines.

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image

Figure 7.  Expanded short-range 13C–1H (HSQC) spectra of acetylated enzyme lignin isolated from PrCCoAOMT-deficient Pradiata TEs and synthetic lignins (DHPs), highlighting peaks for benzodiocane units (J). (a) Differential spectrum for line pLTK4-26 versus wild-type control; (b) differential spectrum for line pLTK4-35 versus wild-type control; (c) spectrum for DHP from a mixture of caffeoyl alcohol and coniferyl alcohol (20:80); (d) spectrum for DHP from a mixture of methyl caffeate and coniferyl alcohol (20:80).

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusion
  7. Experimental procedures
  8. Acknowledgements
  9. References
  10. Supporting Information

Effect of PrCCoAOMT suppression on lignin content

Suppression of genes associated with the monolignol pathway in pine, such as 4CL and HCT (encoding 4-coumarate CoA ligase and p-hydroxycinnamoyl-CoA shikimate hydroxycinnamoyltransferase), resulted in substantial lignin reductions of up to 50–60% (Wagner et al., 2007, 2009). In comparison, PrCCoAOMT suppression caused only quite moderate reductions in lignin content of 5–20%, and despite an up to 90% reduction in PrCCoAOMT steady-state RNA levels in transgenic lines. A possible explanation for this phenotypic discrepancy may be that methyltransferases other than CCoAOMT support the methylation of caffeoyl-CoA in pine. In vitro studies indicated that both CCoAOMT and COMT are capable of methylating caffeoyl-CoA in angiosperms (Do et al., 2007). Conifers do not synthesize S-type lignin, for which COMT is required in angiosperms (Rastogi and Dwivedi, 2008). However, conifers contain a multi-functional methyltransferase called AEOMT (SAM:hydroxycinnamic acids/hydroxycinnamoyl-CoA esters O-methyltransferase), which also supports methylation of caffeoyl-CoA in vitro (Li et al., 1997). In addition, methyltransferases encoded by COMT-like genes with unknown function or CCoAOMT-like genes expressed in the developing xylem could also potentially methylate caffeoyl-CoA, thus preventing greater reductions in lignin content (Friedmann et al., 2007; Koutaniemi et al., 2007).

Effects of PrCCoAOMT suppression on lignin composition and structure

In addition to reducing lignin levels, PrCCoAOMT suppression also affected lignin composition in TEs of PrCCoAOMT-deficient lines. Both pyrolysis GC/MS and NMR spectra indicated a reduction of G-type compared to H-type lignin (Figures 4 and 5). An up to approximately 10-fold increase in the H/G ratio in transgenic line pLTK4-26 compared to the wild-type control was identified by comparing the corresponding 2D 13C–1H correlation (HSQC) spectra of the aromatic region (Table 2). The magnitude of this change was dependent on the degree of PrCCoAOMT suppression, as line pLTK4-35 showed less dramatic changes than pLTK4-26. The observed rise in the H/G ratio is consistent with the role of CCoAOMT in the monolignol pathway (Figure 8), and confirms the presumed function of this enzyme in the biosynthesis of G-type lignin in pine. By comparison, HCT suppression caused an up to 50-fold increase in the H/G ratio in HCT-deficient TEs (Wagner et al., 2007). HCT represents the first committed metabolic step in the biosynthesis of G-type lignin in pine (Figure 8) (Wagner et al., 2007), and this may explain why HCT suppression had a stronger effect on the H/G ratio than suppression of PrCCoAOMT. This result is consistent with the situation in angiosperm species, in which large increases in H-type lignin were only observed when enzymes at the start of the G/S-type specific pathway were suppressed (Reddy et al., 2005; Chen et al., 2006; Shadle et al., 2007).

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Figure 8.  Proposed biosynthesis of p-hydroxyphenyl-, catechyl- and guaiacyl-type monolignols in PrCCoAOMT-deficient Pradiata lines, starting from phenylalanine. PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate-CoA ligase; HCT, p-hydroxycinnamoyl-CoA shikimate hydroxycinnamoyltransferase; C3H, p-coumarate 3-hydroxylase; CCoAOMT, caffeoyl-CoA O-methyltransferase; CCR, cinnamoyl-CoA reductase; CAD, cinnamyl alcohol dehydrogenase.

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The quantities of the inter-unit linkages (A), (B) and (C) (Figure 6) detected in short-range 13C–1H (HSQC) spectra were comparable between PrCCoAOMT-deficient lines pLTK4-26 and pLTK4-35 and the wild-type control. The slight decrease of (D) in PrCCoAOMT-deficient lines may be the consequence of reduced coniferyl alcohol levels in transgenic lines, a trend previously observed in HCT RNAi transgenic lines in pine (Wagner et al., 2007). Quantities of cinnamyl alcohol end-groups (X1) were comparable between PrCCoAOMT-deficient lines and the wild-type control. However, this correlation peak may not be purely attributable to this end-group (Stewart et al., 2009; Kim and Ralph, 2010).

A comparison of the short-range 13C–1H (HSQC) spectra from DHPs containing caffeate or caffeyl alcohol with those from PrCCoAOMT RNAi transgenic lines strongly suggested that the presence of benzodioxanes (J) in transgenic lines resulted from incorporation of caffeyl alcohol into the lignin polymer (Figures 6 and 7). This phenotype, which created a lignin polymer consisting of p-hydroxyphenyl (H), catechyl (C) and guaiacyl (G) units, is likely to be a consequence of the position of CCoAOMT in the monolignol pathway (Figure 8). Based on the level of inter-unit linkage (J) in PrCCoAOMT-deficient lines pLTK4-26 and pLTK4-35 (Table 2), caffeyl alcohol represents a minor lignin constituent. However, a higher caffeyl alcohol level may be present in transgenic line pLTK4-22, which displayed the strongest phenotype in pyrolysis-GC/MS (Figure 4) and ABSL lignin assays (Table 1). Our results imply that, in pine, cinnamoyl-CoA reductase and cinnamyl alcohol dehydrogenase are capable of converting caffeoyl-CoA to caffealdehyde and caffeyl alcohol, respectively (Figure 8). The enzyme kinetics of these reactions remain to be determined.

The incorporation of caffeyl alcohol into the lignin polymer in pine may be compared with incorporation of 5-hydroxyconiferyl alcohol into lignin in COMT-deficient angiosperm species (Marita et al., 2003; Morreel et al., 2004; Lu et al., 2010). Both cases involve suppression of methyltransferases (CCoAOMT and COMT) that are required for methylating monolignol intermediates. In both cases, decreased methyltransferase activity resulted in formation of monomers/monolignols with an ortho-diphenol structure, which participate in the formation of benzodioxane units in lignin (Morreel et al., 2004; Lu et al., 2010; this study). In contrast to pine, suppression of CCoAOMT in angiosperm species such as Arabidopsis, alfalfa (Medicago sativa), poplar (Populus tremula × Populus alba) and tobacco (Nicotiana tabacum) did not result in incorporation of caffeyl alcohol and the concomitant production of benzodioxanes in the lignin polymer (Zhong et al., 1998, 2000; Meyermans et al., 2000; Marita et al., 2003; Do et al., 2007). A number of factors could have interfered with the incorporation of caffeyl alcohol in the lignin polymer in angiosperms. For example, ortho-diphenols such as caffeyl alcohol are highly reactive and likely to be subject to oxidation by polyphenol oxidases and catechol dioxygenases (Weng and Chapple, 2010). Also, caffeyl alcohol could form quinones, which interfere with the radical coupling reactions required for incorporation of monolignols into the lignin polymer (Grabber et al., 2010). In addition, caffeoyl CoA has the potential to be channeled into pathways such as flavonoid biosynthesis (Morreel et al., 2006), which could have compromised the production of caffeyl alcohol.

Conclusion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusion
  7. Experimental procedures
  8. Acknowledgements
  9. References
  10. Supporting Information

This study on CCoAOMT suppression in pine establishes CCoAOMT as an enzyme that is required for biosynthesis of guaiacyl lignin in gymnosperms. PrCCoAOMT suppression affected lignin content and resulted in a lignin polymer with an unusual subunit composition. In particular, this study provides evidence for caffeyl alcohol incorporation into lignins, and therefore a role for caffeyl alcohol as a possible lignin monomer. These changes indicate a metabolic plasticity in lignification in pine that is consistent with the existing theory that monolignols are cross-coupled onto the growing polymer in a chemically controlled fashion, independently of enzymes or other proteins (Ralph et al., 2004). The metabolic plasticity of the lignification process allows this polymer to vary substantially in composition and structure, providing avenues for development of improved feedstocks for biomaterials and biomass conversion processes. Finally, this study confirmed that the pine TE system is an excellent experimental platform for lignin manipulation to improve our understanding of the lignification process in conifers.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusion
  7. Experimental procedures
  8. Acknowledgements
  9. References
  10. Supporting Information

Clone isolation and construct design

A 792 bp PCR fragment of a P. radiata CCoAOMT cDNA clone containing the 780 bp open reading frame was isolated from a P. radiata cDNA library derived from differentiating xylem. The primers CCoAOMT61 (5′-AACTAGTTTAACGAAATGGCAAGC-3′) and CCoAOMT842 (5′-AGGACTAGTTTCAATAGACACGCC-3′) were designed based on pre-existing sequence information from Pinus taeda CCoAOMT clone AF036095 (Li et al., 1999). The amplified PCR fragment of the putative PrCCoAOMT cDNA clone was inserted into pGEM-T Easy (Promega, http://www.promega.com/), sequenced and subsequently cloned in the sense and antisense orientations into a derivative of pAHC25 (Christensen et al., 1992). The resulting plasmid containing the final PrCCoAOMT RNAi construct was named pLTK4 (Figure 2).

Tissue culture, transformation and molecular monitoring procedures

Xylogenic Pradiata callus cultures were co-transformed with plasmids pLTK4 and pAW16 (Figure 2) (Wagner et al., 2007) as described previously (Möller et al., 2003). Integration of the PrCCoAOMT RNAi construct into the genome was monitored by genomic PCR using the primer pairs M13fwd (5′-CCCAGTCACGACGTTGTAAAACG-3′) and CCoAfwd (5′-GCAGATCTGTTTAACGAAATGGCAAGCACAAG-3′), and CCoAfwd (as above) and NOS-terrev2 (5′-ATTGCCAAATGTTTGAACGA-3′). Expression of PrCCoAOMT in transgenic and non-transformed cell lines was monitored by quantitative RT-PCR as described previously (Cato et al., 2006) using primers Tq26TT54_200_06_F1 (5′-TTGCAGGCGTGTCTATTGAAAACAATC-3′) and Tq26TT54_200_06_R1 (5′-CAAATGGCTTCAACCCCATA-3′).

Microscopic analysis of pine callus cultures

Pradiata callus cultures were maintained and induced to differentiate tracheary elements (TEs) as described previously (Möller et al., 2006). Cultures freshly induced to form TEs were stained with Congo red (0.1% in 50% ethanol) for 10 min, washed in water for 1 min, then stained with acriflavin (0.0025% in water) for 10 min and mounted in 50% glycerol. Tissue was imaged using a Leica TCS NT confocal microscope (http://www.leica.com/) using 488/568 nm excitation and 530/600 nm emission.

Chemical fingerprinting of pine callus cultures

Pyrolysis-GC/MS was performed on differentiated, powdered and freeze-dried pine callus cultures and purified TEs as described previously (Möller et al., 2003; Wagner et al., 2007). Thermal breakdown products of pyrolysed materials were identified using mass spectra of lignin and polysaccharide-derived pyrolysis products (Faix et al., 1990, 1991a,b; Ralph and Hatfield, 1991).

Preparation of cellulolytic enzyme lignins from purified TEs

Preparation of whole cell-wall and cellulolytic enzyme lignin samples for NMR was performed essentially as described previously (Lu and Ralph, 2003; Wagner et al., 2007). In brief, isolated TEs were extracted with 80% aqueous ethanol (sonication, 3 × 20 min). Isolated cell walls (151.2, 240.8, and 247.8 mg for wild-type, pLTK4-26 and pLTK4-35) were ball-milled (3 × 20 min, 5 min cooling cycle) using a Retsch PM100 ball mill (http://www.retsch.com/) vibrating at 600 rpm with ZrO2 vessels containing ZrO2 ball-bearings (Kim and Ralph, 2010). The ball-milled walls were transferred to centrifuge tubes and digested at 30°C using crude cellulases (Cellulysin; Calbiochem, http://www.calbiochem.com; lot number D00074989; 30 mg g−1 of sample, in pH 5.0 acetate buffer; three times over 2 days; fresh buffer and enzyme added each time), leaving all of the lignin and residual polysaccharides totalling 47.8 mg (39% of the original cell wall, wild-type), 32.3 mg (26%, pLTK4-26) and 51 mg (40%, pLTK4-35) (Lu and Ralph, 2003; Wagner et al., 2007). For NMR characterization, the cellulase-digested cell walls were subjected to solubilization and acetylation in DMSO/N-methylimidazole/acetic anhydride (Lu and Ralph, 2003) to produce 70.5, 48.1 and 78.6 mg of acetylated product, respectively.

Lignin quantification

The amount of acetyl bromide-soluble lignin (ABSL) in purified TEs from transgenic and non-transformed cell lines was determined as described previously (Wagner et al., 2007).

Generation of dehydrogenation polymers (DHPs)

Three solutions were prepared for DHP synthesis. To produce solution A, caffeyl alcohol (33.2 mg, 0.2 mmol) or methyl caffeate (33.8 mg, 0.2 mmol) and coniferyl alcohol (144.2 mg, 0.8 mmol) were dissolved in acetone (24 ml), and the solution was added to sodium phosphate buffer (216 ml, 0.1 m, pH 6.5). Solution B comprised hydrogen peroxide aqueous solution (240 ml, 0.005 mmol ml−1). For solution C, horseradish peroxidase (5 mg, type II, 188 units mg−1; Sigma, http://www.sigmaaldrich.com/) was dissolved in 60 ml of sodium phosphate buffer. Solutions A and B were added dropwise to solution C at 25°C over a period of 18 h at a constant flow rate using a peristaltic pump. The reaction mixtures were stirred for a further 6 h. The resulting DHP precipitate was collected by centrifugation (10 000 g, r.t., 15 min), re-suspended in ultra-pure water (100 ml) at r.t. for 5 min and then recovered by centrifugation (10 000 g, r.t., 15 min). This operation was repeated three times and the final pellets were lyophilized producing DHPs as a white powder (111.3 mg from caffeyl alcohol and 111.1 mg from methyl caffeate). For NMR analysis, the DHPs were acetylated in pyridine/acetic anhydride using a standard protocol (Brunow and Lundquist, 1991; Cathala et al., 2003).

NMR spectroscopy

The NMR methods used have been described previously (Lu and Ralph, 2003; Wagner et al., 2007), with the exception that soft-180-adiabatic-pulse variants of the HSQC experiments (Kupče and Freeman, 2007; Hedenström et al., 2009; Kim and Ralph, 2010) were used. Such experiments are less sensitive to differences in 1-bond 13C–1H coupling constants, and the response over the entire spectral range is more uniform, suggesting that improved quantification should result. NMR spectra were acquired on a Bruker Biospin AVANCE 500 MHz spectrometer (http://www.bruker.com/) fitted with a cryogenically cooled 5 mm triple resonance probe (TCI) with inverse geometry (proton coils closest to the sample). Acetylated cellulolytic enzyme lignins isolated from TEs and acetylated DHPs (20–40 mg) were dissolved in 0.5 ml of chloroform-d, and the central chloroform solvent peak was used as an internal reference (δC, 77.0; δH, 7.26 ppm). HSQC experiments were performed using the following parameters: acquired from 10 to 0 ppm in F2 (1H) with 1998 data points (acquisition time 200 msec), and from 200 to 0 ppm in F1 (13C) with 400 increments (F1 acquisition time 8 msec) of 30 scans with a 1.5 sec inter-scan delay; the d24 delay was set to 0.89 msec (1/8J, J: 140 Hz). Processing used typical matched Gaussian apodization in F2, and squared cosine-bell and one level of linear prediction (32 coefficients) in F1. Volume integration of contours in HSQC plots (Wagner et al., 2007) was performed using Bruker’s TopSpin 2.1 software and no correction factor was used. For quantification of H/G distributions, only the carbon-2 correlations from G units and the carbon-2/6 correlation from H units were used, and the G integrals were doubled. For a rough estimation of the various inter-unit linkage types, the following well-resolved contours were integrated: Aα, Bα, Cα, Dα, Jβ and X1. However, X1 is not included in the total, which reflects only the inter-unit linkages: the percentage of X1 in Table 2 is expressed as a percentage of the total inter-units (AJ).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusion
  7. Experimental procedures
  8. Acknowledgements
  9. References
  10. Supporting Information

This work was funded in part by grants C04X0207 and C04X0703 from the New Zealand Foundation for Research, Science and Technology. J.R. was funded in part by the US Department of Energy Great Lakes Bioenergy Research Center (Department of Energy Office of Science BER DE-FC02-07ER64494). We would like to thank Tim Strabala and Brian Richardson for critical reading of this manuscript.

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  3. Introduction
  4. Results
  5. Discussion
  6. Conclusion
  7. Experimental procedures
  8. Acknowledgements
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusion
  7. Experimental procedures
  8. Acknowledgements
  9. References
  10. Supporting Information

Figure S1. Alignment of the deduced amino acid sequence of the isolated P.    radiata CCoAOMT clone PrCCoAOMT (a; GenBank: HQ444753) with putative orthologs from Pinus taeda (b; GenBank: AF036095), Picea abies (c; GenBank: CAK18782.1), Picea glauca (d; GenBank: GQ03207_P07), and Picea sitchensis (e; GenBank: WS02736_J21) using ClustalW. Amino acids that differ from the isolated PrCCoAOMT clone are shown in line b–e.

Figure S2. Mechanistic scheme for the production of benzodioxanes 6 in lignins via incorporation of caffeyl alcohol 1C into a predominantly guaiacyl lignin. Only the pathways producing β-ether units are shown, but β-5- (phenylcoumaran) units are also possible. Cross-coupling of caffeyl alcohol 1G, via its radical 1G., into the growing polymer, shown here with a guaiacyl phenolic endgroup (2G), produces the usual quinone methide 3 which is rearomatized by water addition to produce a 3,4-dihydroxyphenyl end-unit 4 (generically a lignin unit 2C). Addition of a new monomer, here coniferyl alcohol 1G (via its radical 1G.) to this end-unit again produces a quinone methide 5. Quinone methide 5, however, has a new pathway for rearomatization – it is internally trapped by the unusual 3-OH in these caffeyl-derived units, producing benzodioxane 6 which can be further incorporated into the lignin polymer by cross-coupling at the 4-O- or 5-positions indicated by the dotted arrows. Cross-coupling of the 3,4-dihydroxyphenyl end-unit in 2C with another caffeyl alcohol monomer 1C is also possible but must be less pronounced in lignins which have only a low caffeyl alcohol-derived content. G) guaiacyl unit; C) caffeyl unit.

Table S1. Measurement of PrCCoAOMT steady state mRNA levels in transgenic lines pLTK4-22, pLTK4-26 and pLTK4-35 using quantitative RT-PCR.

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