This Article Abstract Full Text (PDF) Supplemental Data All Versions of this Article: 19/11/3669    most recent tpc.107.054148v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via CrossRef Citing Articles via ISI Web of Science (5) Citing Articles via Google Scholar Google Scholar Articles by Leplé, J.-C. Articles by Boerjan, W. Search for Related Content PubMed PubMed Citation Articles by Leplé, J.-C. Articles by Boerjan, W. Agricola Articles by Leplé, J.-C. Articles by Boerjan, W.

Downregulation of Cinnamoyl-Coenzyme A Reductase in Poplar: Multiple-Level Phenotyping Reveals Effects on Cell Wall Polymer Metabolism and Structure^[W]

^a Department of Plant Systems Biology, Flanders Institute for Biotechnology, 9052 Gent, Belgium
^b Department of Molecular Genetics, Ghent University, 9052 Gent, Belgium
^c Unité Amélioration Génétique et Physiologie Forestières, Institut National de la Recherche Agronomique, 45166 Olivet cedex, France
^d Unité de Chimie Biologique, Unité Mixte de Recherche 206 AgroParisTech/Institut National de la Recherche Agronomique, AgroParisTech Centre de Grignon, 78850 Thiverval-Grignon, France
^e Institut fur Forstbotanik, Universität Göttingen, 37077 Göttingen, Germany
^f Department of Wood Science, University of British Columbia, Vancouver, BC, Canada V6T 1Z4
^g U.S. Dairy Forage Research Center, Agricultural Research Service, Department of Horticulture, University of Wisconsin, Madison, Wisconsin 53706
^h Centre de Recherche sur les Macromolécules Végétales, Unité Propre de Recherche 5301, Centre National de la Recherche Scientifique, 38041 Grenoble Cedex 09, France
ⁱ Pôle de Biotechnologies Végétales, Unité Mixte de Recherche/Unité Propre de Service 5546, Centre National de la Recherche Scientifique, 31326 Castanet Tolosan, France
^j Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, 90183 Umeå, Sweden
^k Max-Planck Institute of Molecular Plant Physiology, 14476 Golm-Potsdam, Germany
^l Centre Technique du Papier, 38044 Grenoble Cedex 9, France
^m U.S. Dairy Forage Research Center, Agricultural Research Service, U.S. Department of Agriculture and Department of Biological Systems Engineering, University of Wisconsin, Madison, Wisconsin 53706

³ Address correspondence to wout.boerjan{at}psb.ugent.be.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
Cinnamoyl-CoA reductase (CCR) catalyzes the penultimate step in monolignol biosynthesis. We show that downregulation of CCR in transgenic poplar (Populus tremula x Populus alba) was associated with up to 50% reduced lignin content and an orange-brown, often patchy, coloration of the outer xylem. Thioacidolysis, nuclear magnetic resonance (NMR), immunocytochemistry of lignin epitopes, and oligolignol profiling indicated that lignin was relatively more reduced in syringyl than in guaiacyl units. The cohesion of the walls was affected, particularly at sites that are generally richer in syringyl units in wild-type poplar. Ferulic acid was incorporated into the lignin via ether bonds, as evidenced independently by thioacidolysis and by NMR. A synthetic lignin incorporating ferulic acid had a red-brown coloration, suggesting that the xylem coloration was due to the presence of ferulic acid during lignification. Elevated ferulic acid levels were also observed in the form of esters. Transcript and metabolite profiling were used as comprehensive phenotyping tools to investigate how CCR downregulation impacted metabolism and the biosynthesis of other cell wall polymers. Both methods suggested reduced biosynthesis and increased breakdown or remodeling of noncellulosic cell wall polymers, which was further supported by Fourier transform infrared spectroscopy and wet chemistry analysis. The reduced levels of lignin and hemicellulose were associated with an increased proportion of cellulose. Furthermore, the transcript and metabolite profiling data pointed toward a stress response induced by the altered cell wall structure. Finally, chemical pulping of wood derived from 5-year-old, field-grown transgenic lines revealed improved pulping characteristics, but growth was affected in all transgenic lines tested.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
Lignins are defined as complex, heterogeneous polymers of 4-hydroxy-phenylpropanoid units (Boerjan et al., 2003; Ralph et al., 2004, 2007a). They are present mainly in the walls of secondary-thickened cells of vascular plants and represent 20 to 30% of the dry weight of wood. Lignins confer rigidity to the cell wall for structural support and impermeability for transport of water and nutrients over large distances. The intrinsic properties of the lignin polymers have been essential for plants to adapt to a terrestrial habitat, enabling them to grow upward, but are also crucial in determining the value of plants as raw materials. For example, lignins are a major concern for the pulp and paper industry because they need to be extracted from the wood by harsh chemical conditions to produce pure cellulose fibers (Peter et al., 2007). Similarly, they are the main limiting factor in fodder digestibility and in the conversion of plant biomass to fermentable sugars in the process to bioethanol (Chen and Dixon, 2007). Over the past decade, considerable attention has been focused on understanding the lignin biosynthetic pathway and on exploring the potential of genetic engineering to tailor lignin content and composition for industrial applications (Baucher et al., 2003; Boudet et al., 2003).

Although the roles of most genes of the monolignol pathway in determining lignin amount and composition have been elucidated, our knowledge is still scarce on how monolignol biosynthesis integrates into wider plant metabolism and how plant metabolism responds to changes in the expression of individual monolignol biosynthesis genes. With the advent of genomic tools that enable unbiased transcriptome- and metabolome-wide analyses, such interactions can now be elucidated. Indeed, deep phenotyping of transgenic plants defective in monolignol biosynthesis has revealed far-reaching consequences on gene expression in various pathways (Ranjan et al., 2004; Rohde et al., 2004; Robinson et al., 2005; Abdulrazzak et al., 2006; Shi et al., 2006; Dauwe et al., 2007). Knowledge of these broader effects at the transcriptome and metabolome levels is essential to fully comprehend the relationships between gene function and cell wall properties, how these cell wall properties are elaborated, and how they relate to the quality of raw material destined for agroindustrial processes (http://www.epobio.net/).

Cinnamoyl-CoA reductase (CCR; EC 1.2.1.44) catalyzes the conversion of feruloyl-CoA to coniferaldehyde and is considered the first enzyme in the monolignol-specific branch of the phenylpropanoid pathway (Lacombe et al., 1997). Because downregulation of the CCR gene in annual model plants significantly reduced lignin content (Piquemal et al., 1998; Chabannes et al., 2001a, 2001b; Jones et al., 2001; Pinçon et al., 2001; Goujon et al., 2003), downregulating CCR in a woody perennial was an interesting potential avenue to improve wood quality for pulping. Here, we investigated the consequences of altering CCR expression in transgenic poplar (Populus tremula x Populus alba) at multiple levels.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
Generation of Transgenic Poplars Downregulated for CCR
Previously, we cloned a full-length CCR cDNA from a xylem cDNA library of poplar (Populus trichocarpa cv Trichobel; Leplé et al., 1998). BLAST alignments against the P. trichocarpa cv Nisqually 1 genome sequence (Tuskan et al., 2006) indicated the presence of a single gene model corresponding to this cDNA, whereas seven additional CCR homologous genes are present in the poplar genome. The CCR gene we cloned is the only one that is strongly expressed in developing poplar xylem (Li et al., 2005).

The poplar CCR cDNA sequence was used to design sense and antisense constructs under the control of the cauliflower mosaic virus (CaMV) 35S promoter for downregulation of the CCR expression. Following the introduction of four different constructs into poplar (P. tremula x P. alba), 40 to 60 independent transformants were regenerated for each construct. Approximately 5% of all transformants, either from sense or antisense lines, was dwarfed. These plants could be maintained for up to 7 months in tissue culture but died upon in vitro propagation and acclimation steps. The remainder of the lines had no apparent growth retardation in the greenhouse. To identify lines that were downregulated for CCR, all transformants were screened for the presence of an orange-brown coloration of the xylem after 3 and 7 months of growth in the greenhouse. This phenotype was previously observed in transgenic tobacco (Nicotiana tabacum) severely depressed for CCR activity (Piquemal et al., 1998). Based on this screen, five transgenic lines displaying the xylem coloration were identified: two (FAS13 and FAS18) and three (FS3, FS30, and FS40) lines transformed with the antisense and the sense construct, respectively (Figure 1A ). RT-PCR indicated reduced steady state CCR transcript levels down to 3 to 4% of wild-type levels (see Supplemental Table 1 online). Four of these transgenic lines (FS3, FS30, FAS13, and FAS18) were selected in 1999 to be field-grown for up to 8 years (Figure 1D).

View larger version (92K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Phenotype of CCR-Downregulated Plants. (A) Basal part of debarked stems of 4-month-old wild-type and CCR-downregulated poplars (FAS13 and FS3). (B) Occasional orange-brown coloration in patches along the stem. (C) Cross section through a branch of a 7-year-old field-grown CCR-downregulated poplar transformant (FAS13). The orange-brown coloration is absent in tension wood. TW, tension wood; OW, opposite wood. (D) Field trial of CCR-downregulated and wild-type poplars.

For the trees grown in the field trial, the xylem coloration was consistently absent from the upper side of the branches (tension wood zone), whereas it was present on the opposite wood side (Figure 1C). Furthermore, the coloration was generally more pronounced in the basal part of the branches and the stem, whereas it became mottled and ultimately disappeared toward the apical end.

Because the intensity of the xylem coloration often varied among lines and among ramets of a given line and because the molecular phenotype correlated with the color intensity (see below), particular lines were preferred in one experiment and other lines in other experiments, depending on the xylem color intensity these trees presented at the harvest time for the different experiments. For particular experiments, red and white patches of the same stem were compared for phenotypic consequences of CCR downregulation.

Histochemical and Autofluorescence Changes Associated with CCR Downregulation
Patchy stems of 6-month-old greenhouse-grown plants and patchy branches of trees from the field trial were cross-sectioned and analyzed for overall morphology, altered lignification by Wiesner (phloroglucinol-HCl) and Mäule staining, altered cellulose content by Astra Blue staining, and for autofluorescence upon excitation with long-wavelength UV and blue light (Figure 2 ; see Supplemental Figure 1 online). Phloroglucinol-HCl and Mäule staining are considered to stain specifically cinnamaldehyde end groups (Adler et al., 1948) and syringyl (S) units in lignin (Lewis and Yamamoto, 1990), respectively. Under long-wavelength UV light, at elevated pH (pH 10.3), ferulate esters fluoresce intensely green (Harris and Hartley, 1976).

View larger version (119K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Histochemical Changes Associated with the Orange-Brown Xylem of CCR-Downregulated Poplar. (A) Blue-excited autofluorescence of a stem section of a 6-month-old greenhouse-grown CCR-downregulated poplar (FAS13) with patchy orange-brown xylem coloration. Blue-excited autofluorescence (450 to 490 nm) shown for details ([a] and [c]) of the cross section (b). The autofluorescence was increased preferentially in the vessels and the middle lamella and/or S1 layers of fibers in the orange-brown xylem zone. The whitish xylem areas of the transformants had no or weak blue-excited autofluorescence, reminiscent of the wild type (data not shown). Exposure time was 1 s. (B) Astra blue and phloroglucinol staining. The cross section from (A) (panel [b]) was stained with phloroglucinol (b) or with both phloroglucinol and Astra Blue ([a] and [c]). (d) and (e) are cross sections through a wild-type branch (d) and an orange-brown zone in a FAS13 branch (e) from field-grown poplars, stained with phloroglucinol. Astra Blue staining was more intense, whereas phloroglucinol staining was less intense in the orange-brown zones of the transformants compared with the wild type.

Together, these data suggest that CCR downregulation reduces the level of hydroxycinnamaldehydes and S units in lignin, increases the level of ferulate esters in lignin, and increases cellulose content or its accessibility by Astra Blue. Furthermore, the blue light autofluorescence in colored zones suggests the presence of metabolites or cell wall structures that are undetectable in noncolored areas.

Cell Wall Ultrastructural Morphology
Transmission electron microscopy (TEM) of cell walls stained with uranyl acetate depicts the macromolecular arrangement of the cell walls. The pattern of differential staining underscores the subdivision of the secondarily thickened walls in three sublayers, S1, S2, and S3. In poplar fibers, the S3 sublayer is generally not distinguishable. Figure 3 shows that in the colored xylem of the transformants, the inner side of the cell wall of the fibers (sublayer S2) and, more rarely, of the vessels (sublayers S2 and S3) displayed successive concentric sublayers. This stratification ranged from absent to extensive, and in some extreme cases, the cell wall appeared disorganized (Figures 3C to 3E). The disorganization was the strongest in the newly formed inner side of the cell wall, became gradually less apparent in the older layers, and was more obvious and more frequently observed in fibers than in vessels. Notably, the S1 layer of both fibers and vessels did not show any sign of altered ultrastructure. The sublayering phenotype was observed neither in the walls of neighboring colorless areas of the transformants nor in those of wild-type cells.

View larger version (66K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. TEM of Xylem Sections of Wild-Type and CCR-Downregulated Poplars and Immunocytochemical Localization of Lignin Epitopes. (A) and (B) The wild type. (C) and (D) FAS18. (E) FAS13. (A) to (E) The sections are stained with uranyl acetate. Fibers and vessels of the wild type have a smooth appearance, and the layers S1 and S2 of fibers and S1, S2, and S3 of vessels are delineated ([A] and [B]). In fibers, S2 is often divided in a dark outer layer and a lighter inner layer (B). In the orange-brown area of CCR transformants, concentric sublayers are visible in the S2 layer of fibers ([C] to [E]) and in the S2 and S3 layers of vessels (D). Similar results were seen in sections of lines FS3 and FS30. The ultrastructure of the cell walls was most severely affected in the orange-brown zones of line FAS13, where the stratification was often accompanied by a loss of compactness (E). (F) and (G) Immunolabeling of syringyl epitopes with S antibody, performed on stem cross sections of wild-type (F) and FS3 (G) poplars, grown under controlled conditions. S epitopes concentrated in the inner part of S2 of fibers in the wild type, whereas a weaker and more homogeneous distribution of S epitopes was observed in S2 of fibers from orange-brown areas of the transformant. Note: differences in the size of gold particles are due to uneven silver enhancement that modified the diameter of the gold particles, but not their number. F, fibers; V, vessels; R, ray cell. Bars = 1 µm.

Altered Lignin Content in CCR-Downregulated Poplars
To analyze whether CCR downregulation reduces lignin content, as suggested by phloroglucinol-HCl and Mäule staining and cell wall structure analysis, branches were collected from 2-year-old field-grown poplars. Xylem fractions were scraped from the young developing xylem of the colored areas of the transgenic poplars and from the corresponding zones of wild-type plants. Acid-insoluble Klason lignin content in xylem samples from the lines FS3, FAS13, and FAS18 was significantly (probability of the least significant difference [P_LSD] < 0.001) reduced by 47, 23, and 8%, respectively, but not from FS30 (see Supplemental Table 2A online). In agreement, the FS3 line had the most intensely colored xylem phenotype, whereas only a light patchy phenotype was observed in the samples of FS30. Additionally, acid-insoluble Klason lignin contents were measured from noncolored and colored stem xylem areas of 1-year-old greenhouse-grown FS3 and FS40. These data indicated that the decreased lignin content was associated with the coloration (see Supplemental Tables 2 and 3 online).

Altered Lignin Structure in CCR-Downregulated Poplars
The lignins of the FS3 and wild-type branch xylem samples and of the FS3 and FS40 stem xylem samples, described above, were analyzed structurally by thioacidolysis (Lapierre et al., 1999). Thioacidolysis selectively cleaves the β–O–4-ether bonds in the lignin polymer. The β–O–4-linked G and S lignin subunits give rise to specific monomeric degradation products that are quantified and reflect the proportion of G and S units linked by β–O–4-ether bonds. The lignin fraction that is released by thioacidolysis is referred to as the noncondensed fraction, in contrast with the units that involve condensed bonds and are not released as monomers by thioacidolysis. The total yield (S+G) and relative proportion (S/G) of the released thioacidolysis monomers are presented in Supplemental Table 2A online. Lignins from the colored area of the FS3 branches systematically released fewer thioacidolysis S and G monomers per gram of Klason lignin than wild-type lignins (P = 0.046), which is indicative of a higher frequency of condensed bonds in CCR-downregulated lines. The S/G ratio based on thioacidolysis data did not differ significantly from that of the wild type. In addition to the main S and G monomers, lignins from the colored zone of the FS3 branches released 10- to 20-fold higher amounts of a new thioacidolysis product than the control lignins (P < 0.001), in which this compound was recovered only in minor amounts (see Supplemental Tables 2A and 3 online). Elucidation of its structure by electron-impact mass spectroscopy (MS) indicated that this new product consisted of an aromatic G ring with a two-carbon side chain in which the and β carbons are involved in a single thioether (CHR; R = SEt) and two thioethers (CHR₂), respectively. This compound was then designated as G-CHR-CHR₂. The corresponding S analog could also be observed but to a much lower extent (data not shown). Thioacidolysis released also increased amounts of ferulic acid from the FS3 colored branch samples. In the patchy stems of 1-year-old greenhouse-grown FS3 and FS40, higher amounts of the G-CHR-CHR₂ marker were released by thioacidolysis from the colored than from the noncolored areas, indicating that it was associated with the coloration. These changes in the release of thioacidolysis monomers were also confirmed in extractive-free xylem and the corresponding milled-wood extracted lignins (MWELs) of 1-year-old greenhouse-grown FS3 compared with the wild type (see Supplemental Table 2B online). In addition, analysis of the dimers released by thioacidolysis from these extractive-free xylem and MWEL fractions indicated a substantially lower yield of the syringaresinol-derived dimer (see Supplemental Table 2B online), as confirmed by nuclear magnetic resonance (NMR) analysis (see below). Furthermore, the MWEL sample of FS3 released twofold more p-hydroxybenzoate, vanillic acid, and ferulic acid upon alkaline hydrolysis (means ± SE based on technical replicates were 40 ± 2, 1.7 ± 0.1, and 0.6 ± 0.1 and 61 ± 0.7, 4.8 ± 1.0, and 1.5 ± 0.2 µmole/g Klason lignin in the wild type and FS3, respectively). Alkaline hydrolysis breaks mainly ester linkages. The release of ferulate is in agreement with the green autofluorescence of stem sections induced by long-wavelength UV excitation, which has been described to be caused by ferulate esters (see Supplemental Figure 1 online; Harris and Hartley, 1976).

Whereas the S/G ratio of thioacidolysis monomers reflects only the etherified units that can release monomeric products, NMR potentially measures the S/G of the entire lignin. The S/G ratio of FAS13 and FS40 lignin, calculated from NMR data, was significantly lower than that of the wild type (see Supplemental Tables 2C and 3 and Supplemental Figure 2 online). These data are supported by the interunit linkage distributions (see below).

The side chain region only peripherally reflects the changes in the S/G distribution but is rich in detail regarding the types and distribution of interunit bonding patterns present in the lignin fraction. The control lignin heteronuclear single-quantum coherence (HSQC) spectrum (Figure 4A ) is typical of a guaiacyl/syringyl lignin containing some residual polysaccharides (Ralph et al., 1999). The lignin was rich in β-aryl ether units A, with modest amounts of phenylcoumaran B, and resinol C, and traces of spirodienone S units, typical for angiosperm lignins. Resinols C arise usually from sinapyl alcohol dimerization and hence are always significantly higher in angiosperms than in gymnosperms. Finally, the cinnamyl alcohol end groups X1, like the resinols C, arise from monomer-monomer coupling (often involving G units) and are therefore relatively minor.

View larger version (16K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Side Chain Regions of HSQC Spectra from Enzyme Lignins Illustrating Lignin Structural Changes. (A) to (C) HSQC spectra from lignins isolated from the wild type (A) and FAS13 (B). The contour colors in (A) and (B) correspond with the lignin units presented in (C). (D) Difference spectrum (FAS13 – wild type) showing, primarily, the decreased resinol C (negative, blue) levels.

Elucidating the Derivation of the G-CHR-CHR₂ Thioacidolysis Compound
The G-CHR-CHR₂ thioacidolysis product could not be derived from any of the normal monolignol coupling products in the lignin polymer because the involvement of the β carbon in two thioethers revealed that this β carbon was at the oxidation level of an aldehyde. Instead, the increased incorporation of ferulic acid into the lignin, as found by thioacidolysis, suggested that G-CHR-CHR₂ was a specific coupling product of ferulic acid into the polymer. To test whether ferulic acid could produce the precursors from which the thioacidolysis marker derives during free-radical polymerization, low levels (5%) of [8-¹³C]-labeled ferulic acid were introduced into a 50:50 coniferyl alcohol:sinapyl alcohol synthetic lignin (dehydrogenation polymer [DHP]) with peroxidase/H₂O₂ to generate the required radicals. This DHP had the same structure as a DHP without incorporation of ferulic acid but had two small characteristic ferulic acid–derived components as seen by their ¹³C₈–¹H₈ correlations at 103.5/6.0 and 116/6.3 ppm in HSQC spectra of the acetylated DHP (see Supplemental Figure 3 online). Thioacidolysis efficiently liberated the G-CHR-CHR₂ marker compound from this synthetic DHP but not from the control DHP without ferulic acid (data not shown). In addition, HSQC correlation peaks matching those in the DHP were found at low levels in the NMR spectra of lignins isolated from transgenic poplars but not in those of the control (see Supplemental Figure 3 online). Therefore, one or both of these structures in the lignin polymer might be the source of the thioacidolysis G-CHR-CHR₂ marker compound. The ferulic acid–enriched DHP was orange-brown colored as the xylem of CCR-suppressed transformants, suggesting that the coloration is due to the presence of ferulic acid during lignification. A complete description of the elucidation of the marker structure and its derivation from ferulic acid–derived structures is reported elsewhere (Ralph et al., 2007b).

HPLC Analyses of Soluble Phenolics
The increased amount of ferulic acid in the lignin of CCR-downregulated poplars, as shown by thioacidolysis and NMR, or esterified, as indicated by alkaline hydrolysis, pointed to an increased flux through the phenylpropanoid pathway toward ferulic acid. To study this flux change, the different cinnamic acids and cinnamaldehydes present in young developing xylem, scraped from 3-month-old greenhouse-grown wild-type and CCR-downregulated poplars (FS3, FS40, and FAS13), were analyzed by liquid chromatography–mass spectrometry (LC-MS) with selected ion monitoring (Morreel et al., 2004b). The concentrations (mean ± SE) of ferulic acid, sinapic acid, coniferaldehyde, and sinapaldehyde were 75.5 ± 16, 197 ± 40, 28.3 ± 4, and 90.8 ± 14 pmole/mg dry weight in the wild-type poplars and 111 ± 30, 384 ± 140, 29.9 ± 12, and 77.3 ± 29 pmole/mg dry weight in the CCR-downregulated lines, respectively. Because of the large variation, the mean values did not significantly differ between the transgenic lines and the wild type with a nested analysis of variance (ANOVA) model. However, when the six ratios of the ion current signal of these four intermediates were analyzed by the same statistical model (Morreel et al., 2004b), all four cinnamic acid:cinnamaldehyde ratios were approximately doubled in CCR-downregulated lines (Table 1 ), indicating that the concentrations of ferulic and sinapic acids had increased relative to those of coniferaldehyde and sinapaldehyde in the CCR-downregulated lines.

Transcriptome Analysis
The phenotypes of the transgenic lines described above might merely result from an altered flux through monolignol biosynthesis. Alternatively, at least part of these phenotypes might be mediated by transcriptional changes in response to CCR deficiency. To reveal such phenotypic effects at the transcript level, gene expression was compared in the young developing xylem from the stems of 6-month-old greenhouse-grown wild-type poplars and transgenic lines FS3 and FS40. Two pools of xylem material were generated for each line (see Methods) and the transcriptome analyzed through an all-pairwise comparison design in duplicate (see Supplemental Figure 4 online) (Glonek and Solomon, 2004) with a 25K Populus (POP2) microarray (Sterky et al., 2004; www.populus.db.umu.se).

In total, 52 distinct genes were identified whose transcript levels were significantly differential in one or both transgenic lines (see Supplemental Table 5 online). In general, the effect on the transcript levels was stronger in FS3 than in FS40. Strikingly, all 49 genes for which differential transcript levels were revealed in FS40 displayed similar differential transcript levels in FS3. For 32 genes, the transcript levels were increased, whereas for 16 genes, they were decreased in the xylem of both CCR-downregulated lines. For only one gene, the expression was affected in an opposite way in both lines. In FS3, a decreased expression level was found for three additional genes.

Based on the annotations and the functional classification according to the public poplar EST database (POPULUSDB; www.populus.db.umu.se) (Sterky et al., 2004) and additional manual curation, 38 of the 49 common differentially expressed genes (78%) could be grouped into 10 functional categories. For the 11 remaining genes, the function of the most similar Arabidopsis thaliana protein was either unknown (two genes) or no significant similarity with an Arabidopsis protein was found (BLAST score < 100) (nine genes). The complete list of genes with their identification numbers on the microarray, annotation and functional classification, and relative transcript level is given in Supplemental Table 5 online.

Within secondary metabolism, the microarray data confirmed the downregulated expression level of the CCR gene. Furthermore, transcript levels of two distinct genes encoding Phe ammonia lyase (PAL), the enzyme channeling carbon from primary into secondary metabolism via the deamination of Phe, were elevated in the CCR-downregulated poplars. The ammonium liberated by the PAL reaction is reassimilated by Gln synthetase (Croteau et al., 2000). Accordingly, the expression of a Gln synthetase was increased in the transformants.

In addition, the transcript levels of three genes encoding enzymes involved in the metabolism of cell wall matrix polysaccharides (i.e., a myo-inositol oxygenase-like gene [MIOX] and a membrane-bound UDP-D-xylose 4-epimerase [MUR4]) were reduced in the CCR-downregulated lines, whereas that of a (1-4)-β-mannan endohydrolase was elevated (see Supplemental Figure 5 online). The transcript levels of two genes encoding glycosyltransferases, corresponding to the Arabidopsis glycosyltransferase PARVUS (At1g19300) (77 and 80% identity for the two poplar genes), with predicted involvement in pectin biosynthesis (Lao et al., 2003), were decreased in CCR-downregulated lines. These data pointed to a reduced biosynthesis and an increased breakdown or remodeling of hemicellulose and pectin.

Related to cell wall organization, the transcript levels of a putative arabinogalactan protein (AGP) and a lipid transfer protein were decreased. Furthermore, a Ser carboxypeptidase-like gene, likely involved in brassinosteroid (BR) signaling and a Leu-rich repeat receptor-like protein kinase that is similar to the Arabidopsis SERK2 displayed elevated transcript levels in the CCR-downregulated transformants.

The transcript levels of eight genes whose expression levels are typically elevated during stress situations were increased in the CCR-downregulated lines: four genes encoding metallothionein proteins, two genes encoding glutathione S-transferase (GST) (one phi and one tau class GST), a gene encoding an NADP-dependent oxidoreductase similar to -crystallin (ZCr), and a gene encoding a U-box domain protein, similar to the fungal elicitor-induced protein CMPG1 of Petroselinum crispum. Overall, the transcriptome analysis revealed differences in the metabolism of cell wall constituents (lignin, carbohydrates, and proteins) and of stress resistance.

Metabolome Analysis
Complementary to transcript profiling, metabolome analysis can provide profound insight into the effects of gene misregulation on plant metabolism and the molecular mechanisms that provoke a phenotype. Metabolite profiles of young developing xylem of 3-month-old greenhouse-grown wild-type and CCR-downregulated poplars (lines FS3, FAS13, and FS40) were obtained by gas chromatography–mass spectrometry (GC-MS) and subsequently analyzed by principal component analysis and t tests. Of the 802 analyzed compounds, 159 corresponded to known metabolites, of which 20 accumulated differentially in the CCR-downregulated lines compared with the wild type (Table 2 ).

The largest fraction of differentially accumulating metabolites consisted of carbohydrates. In the transgenic poplars, glucose, mannose, galactose, and myo-inositol and the oligosaccharides raffinose and melezitose were all reduced to concentrations 0.5- to 0.8-fold lower than those found in the wild type, reflecting changes in central carbohydrate metabolism. In cell wall polysaccharide metabolism, the concentrations of glucuronate (GlcA) in the transgenic lines were 0.6- to 0.9-fold of those detected in the wild-type poplars, whereas concentrations of xylose and rhamnose were increased 1.8- to 2.6-fold and 1.2- to 1.4-fold, respectively, in the transgenic lines (see Supplemental Figure 5 online). Again, these data point to reduced synthesis and increased breakdown or remodeling of hemicelluloses and/or pectins, assuming that partial degradation of the nucleotide sugars is not at play.

In ascorbate metabolism, decreased levels of myo-inositol, GlcA, and L-gulono-1,4-lactone, a slightly lower concentration of dehydroascorbate dimer (0.8- to 1.0-fold) and 1.9- to 2.2-fold higher levels of glycerate, a possible breakdown product of ascorbate, were detected in the CCR-downregulated lines compared with the wild-type lines (see Supplemental Figure 5 online). Overall, the metabolome analysis revealed a substantial difference in maleate metabolism, modest shifts in carbohydrate metabolism, and a potential effect on ascorbate metabolism (Lorence et al., 2004).

Metabolite Correlation Networks
To reveal additional spots of differential regulation in the metabolism of the transformants, networks of strong correlations (r > 0.80) between metabolite levels were visualized for the wild type and each of the transgenic lines FS3, FS40, and FAS13 (see Supplemental Figure 6 online). In these networks, vertices and edges represent metabolites and strong correlations, respectively. Hubs are those metabolites (vertices) that are strongly correlated to many other metabolites and, therefore, whose synthesis is strongly coregulated with the remainder of metabolism. Four compounds (i.e., mannose 6-phosphate [ID 231001], S-methyl-L-Cys [ID 144002], and two unknown [ID 313003 and 212004]) had many more connections in the correlation networks of the transgenic lines than in those of the wild type, indicating that their synthesis was highly coregulated with many other (primary) metabolites because of the CCR downregulation (see Supplemental Figure 6 and Supplemental Table 6 online). This was most prominent for mannose 6-phosphate that was not found at all in the correlation network of the wild type.

Fourier Transform Infrared Spectroscopy
Whole cell wall chemical changes induced by CCR downregulation were analyzed in stem sections of 6-month-old greenhouse-grown plants by Fourier transform infrared (FTIR) spectroscopy. The differences in absorbance of 15 absorption bands in the fingerprint region between 1800 and 600 cm^–1 of the FTIR spectra (peaks 1 to 15; see Supplemental Figure 7 online) were registered in colored and noncolored areas of xylem sections of transformants (FS3, FS40, and FAS13) and in the wild type. Analyses were done with the ANOVA model 3 that takes the coloration intensity into account to distinguish differences between the poplar lines. In general, significant models were obtained for the intensities of 14 absorption bands (see Supplemental Table 8 online). In addition to data obtained from the literature, FTIR spectra of model compounds were recorded to aid the interpretation of FTIR data from the stem sections.

Cell wall carbohydrates had been previously associated with absorption bands at 1778 to 1691 cm^–1 (1), 1397 to 1349 cm^–1 (7), 1188 to 1145 cm^–1 (10), and 1096 to 999 (12) (see Supplemental Table 8 online). More specifically, the absorption band (1) has been related to ester groups of carbohydrate origin, mainly xylans and pectins in poplar (see Supplemental Table 8 online). The intensity of all four absorption bands decreased with increasing coloration intensity.

FTIR absorption bands solely ascribed to aromatics in the cell wall were observed at 1691 to 1612 cm^–1 (2), 1612 to 1554 cm^–1 (3), and 1527 to 1486 cm^–1 (4). Absorption band (2) is associated with carbonyl groups conjugated with aromatic rings and ferulic acid linked to lignin (see Supplemental Table 8 online). The apex of this absorption band (1652 cm^–1) corresponded to a peak that occurred in the spectrum recorded for free ferulic acid and not in those of four distinct ferulic acid ester references (methyl, ethyl, glucose, and galactose; data not shown). In the spectra of CCR-deficient transformants, as well as in those of the ferulic acid–containing DHP, the intensities of absorption bands (2) and (3) were increased, in agreement with increased ferulic acid incorporation in the wall and in the DHP, and fully correlated with the color intensity of the xylem. Absorption band (4), generally used to quantify lignin (see Supplemental Table 8 online), decreased with increasing coloration.

In summary, FTIR data of the transgenic lines indicated that the cell wall carbohydrate and, even more, lignin content decreased with increasing coloration intensity. The orange-brown color was associated completely with aromatically conjugated carbonyls.

Cell Wall Carbohydrate Analyses
Because both transcriptome and metabolome analyses indicated changes in cell wall polysaccharide metabolism in the transformants, the carbohydrate status of the cell walls in young developing xylem of the stems of 6-month-old greenhouse-grown wild-type and CCR-downregulated FS3 and FS40 poplars was analyzed (Table 3 ; see Supplemental Table 7 online). In the CCR-downregulated transformants, the reduced lignin content was associated with a significant decrease in hemicellulose content, whereas the cellulose content was increased. However, because equal amounts of dry weight were analyzed and the cell wall polymers were measured as percentages, less lignin and hemicellulose will be mass-balanced by cellulose. Hydrolysis of the isolated hemicellulose revealed that the overall hemicellulose composition was similar in wild-type and CCR-downregulated poplar.

The Kappa number, a measure of the residual lignin content in the pulp after cooking, was significantly affected by both the active alkali conditions (P < 0.001) and the poplar line (P = 0.003). As expected, a decrease in the Kappa number (improved delignification) was observed with increasing active alkali in the pulping process (see Supplemental Figure 8A online). At all active alkali charges, the Kappa number of the lines FS3 (P_LSD = 0.012) and FS30 (P_LSD = 0.022) was significantly lower than that of the wild type, whereas that of the FAS13 and FAS18 transgenic lines did not significantly differ from the wild type for any active alkali charge (see Supplemental Figure 8A online).

A high pulp viscosity, reflective of cellulose/hemicellulose degree of polymerization, is generally associated with better pulp and paper properties and is generally lower under higher alkali conditions. Pulp viscosity was significantly lower in the transgenic line FS3 than that in the wild type at all active alkali conditions (P = 0.001; P_LSD = 0.004) (see Supplemental Figure 8B online).

A significant effect of both the active alkali conditions (P < 0.001) and poplar line (P = 0.004) was observed for the proportion of uncooked particles that are complexes of assembled fibers. High levels of uncooked particles indicate that the chemical cooking conditions were not strong enough to totally dissolve the lignin and individualize the fibers. The percentage of uncooked particles was lower under higher alkali conditions and was significantly lower in the transgenic lines FS3 (P_LSD = 0.017) and FS30 (P_LSD = 0.014) than in the wild type for all alkali conditions (see Supplemental Figure 8C online).

Statistical analysis of the screened pulp yield indicated a significant interaction (P = 0.002) between the active alkali conditions and the poplar line. For line FS3, the optimal pulp yield was at 16% (or lower) active alkali, whereas the wild type and the other transformants required 18% active alkali to reach maximum pulp yields (see Supplemental Figure 8D online). One-way ANOVA revealed a significantly lower yield for line FS3 at 18% active alkali (P = 0.04; P_LSD = 0.003).

In summary, the optimal cooking conditions for the wild type were reached at 18% alkali charge. At 16%, the wild-type line had a much higher Kappa number and lower pulp yield than at 18% alkali. Both transgenic lines FS3 and FS30 were more easily cooked at 16% active alkali than the wild type, as revealed by their low percentage of uncooked particles. The two antisense lines FAS13 and FAS18 were not significantly different from the wild-type poplars.

Growth of CCR-Downregulated Poplar in the Field
As described above, downregulation of CCR resulted in stunted plants in 5% of the regenerants, but the lines that were selected for further experiments (FS3, FS30, FS40, FAS13, and FAS18) had an apparently normal development under greenhouse conditions. To investigate whether CCR downregulation impacted the growth characteristics of poplars during further development and under natural circumstances, growth parameters (i.e., the height, girth, girth increase, and volume and volume increase) were determined annually for all trees in the field trial (10 replicates for each of the lines FS3, FS30, FAS13, FAS18, and the wild type) (Figure 5 ). These growth parameters were subjected to a three-way ANOVA involving year, line, and block effects. A three-way interaction was noticed for girth increase, whereas for all other parameters, the full model could be reduced to model 1 that still included a significant interaction between the line and the position of the block in the field. All traits were subject to genotype x environment interaction. The P_LSD values mentioned below are based on the two-way ANOVA model involving the year and line as main factors.

View larger version (18K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Growth Characteristics of Field-Grown CCR-Downregulated Transgenic Poplars. For wild-type and the CCR-downregulated lines FS3, FS30, FAS13, and FAS18, the mean values for height (A), girth (B), girth increase (C), volume (D), and volume increase (E) from 2001 to 2003 are presented. Data are means of 10 biological replicates. Lines for which the given growth characteristic was significantly differential compared with the wild type are marked by an asterisk.

In wild-type poplars, the girth increased linearly from 150 to 270 mm between 2001 and 2003 (Figure 5B). As for height, the values for FAS3 and FAS13 were significantly different (P_LSD < 0.001) from those of the wild type, with 16 and 10% reduction, respectively.

Girth increase was determined in 2002 and 2003 and was on average 57 mm/year in the wild type (Figure 5C). All transgenic lines exhibited a growth reduction between 14 and 23%. These reductions corresponded to 45 mm/year (P_LSD = 0.004), 48 mm/year (P_LSD = 0.04), 44 mm/year (P_LSD = 0.002), and 48 mm/year (P_LSD = 0.03) for the FAS13, FAS18, FS3, and FS30 lines, respectively. Although the slope obtained for line FAS18 was quite different compared with the other lines, no significant clone by year interaction for girth increase was found.

Volume changed exponentially across years and rose from 3550 cm³ in 2001 up to 16,300 cm³ in 2003 in wild-type plants (Figure 5D). In the FAS13 and FS3 lines, a significantly lower volume was found (P_LSD < 0.001 for both lines) with an average reduction of 23 and 43% compared with the wild type, respectively.

In wild-type poplars, the volume increase augmented from 4300 cm³/year in 2002 to 8400 cm³/year in 2003 (Figure 5E). Values for this trait were significantly lower in the transgenic lines FAS13 (P_LSD = 0.001), FS3 (P_LSD < 0.001), and FS30 (P_LSD = 0.04), whose volume increase was on average 31, 51, and 29% lower, respectively, than in the wild type. In conclusion, all transgenic lines evaluated had reduced growth when grown in the field.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
Ferulic Acid Cross-Couples with Lignin
CCR catalyzes the conversion of feruloyl-CoA to coniferaldehyde in the biosynthesis to the monolignols. Downregulation of CCR expression in poplar resulted in a decreased Klason lignin content and reduced abundance of a range of low molecular weight oligolignols, including dimers and trimers of monolignols (see Supplemental Table 4 online) (Morreel et al., 2004a, 2004b), which is in accordance with a decreased monolignol supply in these plants. The reduced lignin content is associated with an orange-brown coloration of the xylem. Sometimes a patchy coloration, correlated with variation in CCR downregulation, was observed along the stem. Patchy phenotypes associated with variation in the efficiency of gene silencing have been observed in several plants, including petunia (Petunia hybrida), where it was first described (Napoli et al., 1990), tobacco (Boerjan et al., 1994), and poplar (Baucher et al., 1996; Tsai et al., 1998; Meyermans et al., 2000; Pilate et al., 2002). In poplar, both developmental and environmental factors seem to contribute to the variability in phenotype (Pilate et al., 2002; this study). These phenotypic variations likely reflect the complexity of the various pathways that trigger and revert gene silencing (Fagard and Vaucheret, 2000; Kanazawa et al., 2007).

It is important to note that the variability in gene silencing can only be observed easily when downregulation of the target gene causes a visible phenotype. When no visible phenotype can be scored, such variability in gene silencing will contribute to biological variation among samples and may obscure reproducibility of phenotypic data.

Transgenic plants deficient in cinnamyl alcohol dehydrogenase (CAD) or caffeic acid 3-O-methyltransferase (COMT) are also typified by a reddish xylem coloration, whose origin has been attributed to the hydroxycinnamaldehydes present during polymerization (Higuchi et al., 1994; Baucher et al., 1996; Tsai et al., 1998; Kim et al., 2000). As CCR catalyzes the biosynthesis of hydroxycinnamaldehydes, the orange-brown coloration in CCR-downregulated plants is probably not caused by such aldehydes. Indeed, phloroglucinol staining that reacts with cinnamaldehyde end-groups in the lignin was strongly reduced in the colored zones, and structural analysis of lignins of the CCR-downregulated transformants indicated that the chemical determinant of the coloration is probably derived from ferulic acid. Indeed, introducing low levels (5%) of ferulic acid into a 50:50 coniferyl alcohol:sinapyl alcohol synthetic lignin (DHP) resulted in a similar coloration of the DHP. It is also noteworthy that semi in vivo incorporation of ferulic acid into tobacco stem cross sections gave a xylem coloration that was similar to the one observed on xylem of tobacco downregulated for CCR (Piquemal et al., 1998).

More direct proof for the incorporation of ferulic acid into lignin was obtained from a range of experiments: thioacidolysis released the G-CHR-CHR₂ structure systematically in higher amounts from lignin of CCR-downregulated poplar than from wild-type lignin (see Supplemental Table 2 online), and the same thioacidolysis marker was released from the synthetic DHP including ferulic acid but not from that without ferulic acid. In addition, two-dimensional NMR spectra of the DHP showed correlation peaks (see Supplemental Figure 3B online) that are characteristic for incorporation of ferulic acid as bis-8–O–4-ethers and simple 4–O–β ethers (Ralph et al., 2007b) and the same correlation peaks were found at low levels in NMR spectra from lignins of the CCR-downregulated transformants but not in those from the wild-type lignin (see Supplemental Figure 3A online). Thioacidolysis also revealed increased amounts of simple 4–O–β-linked ferulic acid, and alkaline hydrolysis indicated an increased amount of esterified ferulic acid, as previously reported in the lignin of CCR-downregulated tobacco (Chabannes et al., 2001a). Further support came from FTIR, absorbances at 1691 to 1612 cm^–1 and 1612 to 1554 cm^–1 in FTIR spectra of the xylem of CCR-downregulated and wild-type trees were positively correlated with each other and with the coloration intensity, indicating an increased amount of aromatically conjugated carbonyls, such as ferulic acids with free carboxyl groups, in the lignin.

Ferulic acid incorporation does not seem to be unique to CCR-downregulated plants, as low levels of G-CHR-CHR₂ were also released by thioacidolysis from wild-type poplar (see Supplemental Table 2 online), as well as tobacco and Arabidopsis, and more substantial levels from grasses (Ralph et al., 2007a). By contrast, a recent study of the Arabidopsis irx4 mutant, deficient in CCR, did not find any evidence for the incorporation of ferulic acid into lignin (Laskar et al., 2006). However, the thioacidolysis and NMR data were not presented in sufficient detail to exclude the incorporation of ferulic acid into lignin. Thus, ferulic acid appears to be incorporated into lignins, particularly in these CCR-deficient transgenic plants and should be considered as an authentic lignin monomer. A more in-depth chemical study of ferulic acid incorporation into lignin, the consequences on lignin structure, and a comparison with the results of Laskar et al. (2006) are presented in Ralph et al. (2007b).

Lignin Structure in CCR-Downregulated Poplar
The increased incorporation of ferulic acid was not the only structural change of the lignins in CCR-downregulated poplar. Indeed, several structural analyses illustrated a relatively stronger effect of CCR downregulation on S units. Thioacidolysis indicated that the lignin from the colored xylem was more condensed and yielded fewer syringaresinol-derived dimers than the wild type (see Supplemental Table 2 online). In agreement with these data, a low S/G ratio and a low frequency of resinol units was found in lignin from the CCR-downregulated lines by NMR (see Supplemental Table 2 online). The thioacidolysis-associated S/G ratio was not statistically different, but, as stated in the results, the latter ratio is based on the uncondensed lignin fraction only, whereas the NMR-associated S/G ratio potentially reflects the whole lignin. In addition, oligolignol profiling demonstrated that the levels of all seven oligolignols detected that involved S units were reduced, whereas the proportions of only one of the five that involved solely G units were reduced in abundance (see Supplemental Table 4 online). These chemical analytical results were supported by the immunolabeling results in TEM, showing a reduced reactivity of the cell walls of the transgenic lines with the S antibody (Figure 3), whereas no obvious variation in labeling intensity was observed with the G antibody (data not shown). The higher frequency of condensed bonds, the lower content of S units involved in β–O–4 or syringaresinol structures, and the higher degree of lignin acylation by p-hydroxybenzoic acid as revealed by alkaline hydrolysis of the MWEL fraction all are reminiscent of early developmental stage poplar lignin (Terashima et al., 1979). In other words, CCR deficiency appears to induce a delay in the lignification program as suggested for the Arabidopsis irx4 mutant by Laskar et al. (2006), which was also characterized by a lower S/G value and a more condensed lignin.

Downregulation of CCR Results in the Accumulation of Phenolic Acid Glucosides
The reduced flux into the monolignol-specific branch of the phenylpropanoid pathway in the xylem of CCR-downregulated poplars results in the strong accumulation of GVA and GSA, of which the latter was not detected at all in wild-type poplars. Interestingly, GVA and GSA also accumulated in CCoAOMT-downregulated poplars, in addition to O³-β-D-glucopyranosyl-caffeic acid (Meyermans et al., 2000; Morreel et al., 2004b). For the CCoAOMT-downregulated poplars, caffeoyl-CoA, the substrate of CCoAOMT, was hypothesized to be redirected to caffeic acid by a thioesterase (Guo et al., 2001). Caffeic acid would then be converted to vanillic and sinapic acid, after which all three acids would be detoxified by glucosylation (Meyermans et al., 2000). In CCR-downregulated poplar, O³-β-D-glucopyranosyl-caffeic acid does not accumulate, but feruloyl-CoA, the substrate for CCR, might be similarly hydrolyzed to ferulic acid. Indeed, as evidenced by thioacidolysis, NMR spectra, and alkaline hydrolysis, lignin of CCR-downregulated poplars contains increased levels of ferulic acid. Subsequently, ferulic acid would be converted to vanillic acid and sinapic acid and further detoxified by glucosylation to GVA and GSA (Meyermans et al., 2000). The weak phloroglucinol staining of the xylem and the increased ratios of either ferulic or sinapic acid to either coniferaldehyde or sinapaldehyde in CCR-downregulated transformants suggest that the reduced activity of CCR results in reduced levels of coniferaldehyde and 5-hydroxyconiferaldehyde. These aldehydes have an inhibitory effect on the COMT- and ferulate-5-hydroxylase–catalyzed conversion of ferulic acid to sinapic acid (Osakabe et al., 1999; Li et al., 2000). Their reduced levels might mitigate this inhibitory effect in CCR-downregulated transformants, facilitating the conversion of ferulic acid to sinapic acid.

In CCR-downregulated tobacco, quinate conjugates of phenylpropanoid intermediates accumulated in addition to glucose conjugates (Dauwe et al., 2007). In tobacco and Arabidopsis, the conversion of p-coumaroyl-CoA to caffeoyl-CoA is thought to occur via quinate ester intermediates (Schoch et al., 2001; Hoffmann et al., 2003), and the enzymes catalyzing this pathway might detoxify accumulating phenylpropanoid intermediates in these species. Remarkably, quinate esters were neither detected in xylem of the wild type nor in that of CCR-downregulated poplars, although the applied HPLC procedure readily allowed their detection in tobacco xylem (Dauwe et al., 2007). Either the quinate esters are present in concentrations below the detection limit or the hexose esters rather than the quinate esters are involved in the transesterification reactions in phenylpropanoid biosynthesis in poplar xylem. p-coumaroyl hexose also has a high group-transfer potential and is detectable in poplar xylem by LC-MS/MS (data not shown).

Taken together, our data indicate that downregulation of CCR results in a decreased flux of feruloyl-CoA to lignin and an increased flux toward ferulic acid, which is detoxified by glucosylation as GSA and GVA, or alternatively is exported to the cell wall where it is cross-coupled with lignin. The increase in ferulic acid levels might simply be the result of a reduced flux to coniferaldehyde, causing a buildup of precursors and derivatives, but possibly ferulic acid biosynthesis might also be induced as a response to a defective cell wall.

Downregulation of CCR Induces PAL Expression
The transcript levels of two PAL genes, encoding the enzyme that catalyzes the entry of Phe into phenylpropanoid metabolism, were increased in the CCR-downregulated poplar lines. One possible reason for the increased PAL transcript levels is that the decreased synthesis of monolignols may signal a need for increased carbon flux into this pathway. A signal for increased developmental lignification could be mediated by mechanistic aspects of the cell wall, resulting from the decreased lignin content, or by the reduced concentrations of pathway intermediates, such as cinnamaldehydes. Alternatively, the disorganization of the cell wall might induce signaling pathways that are typically induced by cell wall damage during wounding or pathogen attack and increase PAL expression. For example, the cellulose synthase mutant cev1 mimics the physiological response characteristic for wounded and infected plants (Ellis et al., 2002). The closest Arabidopsis homolog of both induced poplar PAL genes is At PAL1, and this gene is also induced by pathogen infection and abiotic stress (Raes et al., 2003). Interestingly, in elicitor-treated cell cultures, a strong increase of PAL activity is associated with reinforcement of the cell wall by induced lignin deposition and increased amounts of wall-bound ferulic acid (Hano et al., 2006). Thus, elevated PAL expression is consistent with the hypothesis that ferulic acid deposition in the wall is not simply the result of sequestration of phenylpropanoid intermediates that accumulate because of suppressed CCR activity but is actively induced to strengthen the cell wall.

The suggested wound-like response induced by CCR deficiency is corroborated by the increased abundance of the transcript levels of scavengers of reactive oxygen species (metallothionein) (Mir et al., 2004; Wong et al., 2004) and of enzymes that detoxify oxidative stress metabolites (GSTs and -crystallin-like protein) (Wilce and Parker, 1994; Dixon et al., 2002; Mano et al., 2002). Additionally, transcript levels of a gene encoding a U-box domain protein with a function in ubiquitylation were increased in the transformants, and because this protein is similar to the fungal elicitor-induced protein CMPG1 of P. crispum, the induced expression of this gene supports a stress response. In CCR-downregulated tobacco, transcript and metabolite profiling also reflected oxidative stress. In these plants, the involvement of a wound-like response in the oxidative stress was corroborated by the accumulation of feruloyl tyramine. This compound is typically elicited in solanaceous plants upon wounding or pathogen attack. Alternatively, the molecular stress response in CCR-downregulated tobacco was associated with photooxidative stress caused by an increased efficiency of photosystem II and a concomitantly elevated photorespiration (Dauwe et al., 2007). In the CCR-downregulated poplar lines, the increased transcript levels of a photosystem II reaction center protein and a Gln synthetase similarly support a possible role of photorespiratory H₂O₂ in generating an oxidative stress response.

Altered Hemicellulose and Pectin Metabolism in CCR-Downregulated Transformants
The differential transcriptome and metabolome in CCR-downregulated poplar both pointed toward increased breakdown or remodeling of noncellulosic cell wall polysaccharides (see Supplemental Figure 5 online). In poplar, the major cross-linking cell wall polysaccharides in secondary-thickened cell walls are xylan (18 to 28% of dry weight) and glucomannan (5% of dry weight) (Mellerowicz et al., 2001). The increased transcript level of (1,4)-β-mannan endohydrolase in the transformants suggests an increased breakdown of (gluco)mannan. Furthermore, xylose and rhamnose levels were increased in the transformants. Biochemically, xylose and rhamnose are only liberated during the breakdown of hemicellulose and pectin, mainly xylan and rhamnogalacturonan, respectively. Thus, the accumulation of these metabolites indicates an increased breakdown and/or remodeling of xylan and pectin. On the other hand, the differential transcriptome and metabolome indicated, in a complementary way, a decreased hemicellulose and pectin biosynthesis in the transformants (see Supplemental Figure 5 online). UDP-D-glucuronate (UDP-D-GlcA) is the precursor of most monomers of both pectin and hemicellulose. The synthesis of UDP-D-GlcA is considered rate limi