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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2010 Sep 27;107(41):17803–17808. doi: 10.1073/pnas.1012900107

Distinct cinnamoyl CoA reductases involved in parallel routes to lignin in Medicago truncatula

Rui Zhou a, Lisa Jackson a, Gail Shadle 1, Jin Nakashima a, Stephen Temple b, Fang Chen a, Richard A Dixon a,1
PMCID: PMC2955080  PMID: 20876124

Abstract

Cinnamoyl CoA reductases (CCR) convert hydroxycinnamoyl CoA esters to their corresponding cinnamyl aldehydes in monolignol biosynthesis. We identified two CCR genes in the model legume Medicago truncatula. CCR1 exhibits preference for feruloyl CoA, but CCR2 prefers caffeoyl and 4-coumaroyl CoAs, exhibits sigmoidal kinetics with these substrates, and is substrate-inhibited by feruloyl and sinapoyl CoAs. M. truncatula lines harboring transposon insertions in CCR1 exhibit drastically reduced growth and lignin content, whereas CCR2 knockouts grow normally with moderate reduction in lignin levels. CCR1 fully and CCR2 partially complement the irregular xylem gene 4 CCR mutation of Arabidopsis. The expression of caffeoyl CoA 3-O-methyltransferase (CCoAOMT) is up-regulated in CCR2 knockout lines; conversely, knockout of CCoAOMT up-regulates CCR2. These observations suggest that CCR2 is involved in a route to monolignols in Medicago whereby coniferaldehyde is formed via caffeyl aldehyde which then is 3-O-methylated by caffeic acid O-methyltransferase.

Keywords: lignification, model legume, monolignol, transposon insertion mutagenesis

The pathway for biosynthesis of the guaiacyl (G), syringyl (S), and hydroxyphenyl (H) building blocks of lignin has undergone several revisions. Early models of a metabolic grid (1) were replaced by a more conservative model favoring a specific route through the grid (2). Finally, discovery that 3-hydroxylation of the aromatic ring took place at the level of a shikimate ester (3, 4) resulted in the currently accepted pathway (Fig. S1A), which is linear as far as the formation of feruloyl CoA catalyzed by caffeoyl CoA 3-O-methyltransferase (CCoAOMT). Although this pathway is widely accepted, it is not fully supported by the results of genetic modification to down-regulate specific pathway enzymes (5, 6).

Caffeic acid 3-O-methyltransferase (COMT) is active with caffeyl aldehyde (7), but the model in Fig. S1A designates COMT as primarily responsible for the 5-O-methylation of 5-hydroxyconiferaldehyde (8), consistent with the reduction in S lignin, but not G lignin, in plants with down-regulated COMT expression (5, 9–11). A route via caffeyl aldehyde has been suggested (12, 13), but its physiological relevance is currently unclear, as is whether a specific form of cinnamoyl CoA reductase (CCR) is required for formation of caffeyl aldehyde.

CCR is the first specific committed step in monolignol biosynthesis (14, 15). The Arabidopsis genome possesses 11 annotated CCR homologs (16), two of which, CCR1 and CCR2, encode true CCR enzymes (17). CCRs from poplar (18), Arabidopsis (19), and tomato prefer feruloyl CoA as substrate, with caffeoyl CoA a poor substrate. Here we report the characterization of a CCR from Medicago truncatula with strong preference for caffeoyl CoA and provide evidence for its role in a route to monolignol biosynthesis that by-passes the methylation/reduction steps catalyzed by CCoAOMT and CCR1.

Results

CCR2 from M. truncatula Prefers Caffeoyl and 4-Coumaroyl CoAs.

Blast analysis of M. truncatula-expressed sequence tags with Eucalyptus gunnii CCR (14) as query resulted in identification of at least eight CCR and CCR-like genes, two of which (CCR1 and CCR2, corresponding to tentative consensus numbers 106830 and 100678, respectively, in the Medicago gene index available at http://compbio.dfci.harvard.edu/cgi-bin/tgi/gimain.pl?gudb=medicago) clustered phylogenetically with CCRs previously shown to function in lignin biosynthesis (20). The two proteins share an overall sequence identity of 80.2% and contain the KNWYCYGK motif (Fig. S1B) believed to be critical for catalysis by CCRs (14).

CCR1 and CCR2 were expressed in Escherichia coli, and the purified recombinant enzymes (Fig. S1C) assayed with the four potential substrates (Fig. 1). CCR1 showed the highest turnover number and was most active with feruloyl CoA (Fig. 1C and Table S1). Like CCRs from other plant species (17–19, 21, 22), it exhibited low activity with caffeoyl CoA (Fig. 1A). In contrast, CCR2 preferred caffeoyl and 4-coumaroyl CoAs (Fig. 1 A and B), and its activity with feruloyl and sinapoyl CoAs was strongly inhibited at higher substrate concentrations (Fig. 1 C and D). Curve fitting with Sigmaplot 10 software suggested a sigmoidal response of CCR2 activity to increasing coumaroyl and caffeoyl CoA concentrations, and further kinetic analysis confirmed that CCR2 exhibits positive cooperativity with these substrates, with a Hill coefficient of 1.9–2.0 (Fig. 1 E and F). Based on the calculated catalytic efficiency (Kcat/Km), feruloyl CoA is the preferred substrate for CCR1 (Table S1). Likewise, caffeoyl and coumaroyl CoAs were the preferred substrates for CCR2, at least in vitro.

Fig. 1.

Fig. 1.

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Functional analysis of recombinant Medicago CCRs expressed in E. coli. (A–D) Initial velocity versus substrate concentration curves with caffeoyl CoA (A), coumaroyl CoA (B), feruloyl CoA (C), or sinapoyl CoA (D) as substrates. (E and F) Hill plots for CCR2 with caffeoyl CoA and coumaroyl CoA, respectively, as substrates.

The other Medicago CCR-like genes (20) also were expressed in E. coli, and the recombinant proteins were tested for activity with hydroxycinnamoyl CoA substrates; none was active.

Tissue-Specific Expression of CCR1 and CCR2.

Overall, CCR1 transcripts were expressed at about 10 times the level of CCR2 and were highly expressed in the sixth internode of the stem, whereas CCR2 was highly expressed in the less-mature second internode (Fig. S2 A and B). Mining the Medicago Gene Expression Atlas (23) indicated that both genes were expressed more highly in roots than in stems.

In situ hybridization of cross sections of Medicago stems revealed that both CCR1 and CCR2 are expressed in vascular elements, with weaker expression in the interfascicular (xylem fiber) region. CCR1 and CCR2 exhibited an expression pattern similar to that of CCoAOMT (Fig. S2C).

Genetic Loss-of-Function Analysis of CCR1 and CCR2.

PCR primers from the ORF sequences of CCR1 and CCR2 (Table S2) were used to amplify DNA pools from a population of M. truncatula-Transposon (Nicotiana tabacum) element 1 (Tnt1) retrotransposon insertion lines (24), resulting in identification of two lines with inserts in CCR1 and three lines with inserts in CCR2. NF4532 and NF5145 (referred to as “ccr1-1” and “ccr1-2,” respectively), had Tnt1-retrotransposon inserts in the fourth and first exons of the CCR1 gene, respectively (Fig. 2A), resulting in severe reductions in CCR1 transcript levels (Fig. 2B). ccr1-1 and ccr1-2 homozygotes exhibited stunted growth and did not survive after flowering (Fig. 2C), whereas heterozygotes grew normally. Based on the assumption that only CCR1 activity will be measurable with 50 μM feruloyl CoA, activity assays indicated that CCR1 activity was reduced to less than 15% of the wild-type level in the ccr1 mutant (Fig. 2D).

Fig. 2.

Fig. 2.

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Characterization of retrotransposon insertion lines in Medicago CCR1. (A) Positions of Tnt1 insertions in CCR1. (B) RT-PCR analysis of CCR1 and Actin transcript levels in wild-type and CCR1 insertion lines. (C) Growth of wild-type (WT) Medicago R108 and homozygous ccr1-1 (Left) and ccr1-2 (Right). Plants in the upper left panel were 4 wk postgermination; plants in other panels were 10 wk postgermination. (D) Extractable activities of CCR1 (with feruloyl CoA) in stem extracts from wild-type and ccr1 mutant lines (activity shown as percentage of the average wild-type value). (E) UV autofluorescence (a–c), phloroglucinol staining (d–f), and Maule staining (g–i) of stem cross-sections of wild-type (a, d, and g), ccr1-1 (b, e, and h), and ccr1-2 (c, f, and i). (F) Acetyl bromide lignin levels in internodes 6 and 7 (counting from the top) of stems of wild-type and ccr1-1 and ccr1-2 lines harvested at early flowering. (G) As in F, showing lignin thioacidolysis yields and monomer compositions. All error bars represent SD of three replicates.

Ccr1 knockout mutants exhibited reduced blue lignin autofluorescence and phloroglucinol staining in vascular tissue compared with wild type (Fig. 2E). Mäule staining revealed a dramatic decrease in S lignin in vascular tissue (Fig. 2E). Acetyl bromide lignin was reduced by more than 50% in the ccr1-knockout mutants (Fig. 2F), and thioacidolysis revealed a greater reduction in S than in G monomers (S/G ratio, 0.15–0.19 in the ccr1 mutants compared with 0.29 in wild type) (Fig. 2G).

Lines NF4418, NF7205, and NF10441 (ccr2-1, ccr2-2, and ccr2-3) had Tnt1 insertions in the CCR2 gene (Fig. 3A), resulting in the loss of CCR2 mRNA (Fig. 3B). The mutants had no visible phenotypes under our growth conditions. Because CCR1 activity is only around 7% that of CCR2 activity at 50 μM caffeoyl CoA (Fig. 1A), the extractable activity measured under these conditions should reflect predominantly CCR2 activity. Disruption of CCR2 expression led to a strong decrease in extractable activity toward caffeoyl CoA, whereas activity toward feruloyl CoA increased by 30–60% (Fig. 3C). The CCR2-knockout lines exhibited ≈10% reductions in acetyl bromide lignin and 25% reductions in total thioacidolysis yield (Fig. 3 D and E), with G lignin being more strongly reduced than S lignin (S/G ratio, 0.29–0.33) (Fig. 3E).

Fig. 3.

Fig. 3.

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Characterization of retrotransposon insertion lines in Medicago CCR2. (A) Positions of Tnt1 insertions in CCR2. (B) RT-PCR analysis of CCR2 and Actin transcript levels in wild-type and CCR2 insertion lines. (C) Extractable activities of CCR1 (with feruloyl CoA) and CCR2 (with caffeoyl CoA) in stem extracts from wild-type and ccr2 mutant lines (activity is shown as percentage of average wild-type value). (D) Acetyl bromide lignin levels in internodes 6 and 7 of stems of wild-type, ccr2-1, ccr2-2, and ccr2-3 lines harvested at early flowering. (E) As in D, showing lignin thioacidolysis yields and monomer compositions. Error bars represent SD of three (C) or five (D and E) replicates.

Complementation of Arabidopsis ccr1 Mutants with Medicago CCR1 and CCR2.

To address further the functions of CCR1 and CCR2 in lignin biosynthesis, we tried to complement the Arabidopsis irregular xylem gene 4 (irx4) ccr1 mutant with each of the two Medicago genes under control of the constitutive 35S promoter. The irx4 mutation in ecotype Landsberg erecta (Ler) is a point mutation in the splice site sequence in the second intron of AtCCR1, a gene known to be involved in lignin biosynthesis (19, 25, 26). More than 20 independent transformants were obtained for each transgene, and the expression of Medicago CCR1 or CCR2 was confirmed by RT-PCR with the primers listed in Table S2 (Fig. 4A). Two or three independent transformants with high expression of the Medicago genes were chosen for further analysis. The results obtained with these lines were similar, and representative results from one line are presented here.

Fig. 4.

Fig. 4.

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Complementation of the Arabidopsis irx4 mutant with CCR1 and CCR2. (A) Expression of Medicago CCR1 and CCR2 detected by RT-PCR in transgenic irx4 Arabidopsis. (B) Appearance of plants 20 d postgermination. Upper panel, view from above. Lower panel, side view. (C) Extractable CCR activities in stem extracts from Arabidopsis ecotype Landsberg erecta (Ler), the irx4 mutant, and in irx4 complemented with Medicago CCR1 or CCR2 (activity is shown as percentage of the average of Ler). (D) UV autofluorescence (a–d), phloroglucinol staining (e–h), and Maule staining (i–l) of stem cross-sections of Arabidopsis Col-0 (a, e, and i), irx4 (b, f, and j), and irx4 expressing CCR1 (c, g, and k) or CCR2 (d, h, and l). (E) Acetyl bromide lignin levels in the inflorescence stems of Ler, in the irx4 mutation in the Ler background, and in irx4 mutants complemented with Medicago CCR1 or CCR2, harvested at 25 d postgermination. (F) As above, showing lignin thioacidolysis yields and monomer compositions. All error bars represent the SD of three replicates.

The irx4 mutant exhibits stunted growth (26), with small, spoon-shaped rosette leaves and a short, weak inflorescence (Fig. 4B). The CCR activity toward feruloyl and caffeoyl CoAs was reduced by 94% and 87%, respectively, of that in Ler (Fig. 4C). The total lignin level in the stem also was reduced significantly, based on autofluorescence and histochemical staining with phloroglucinol or Mäule reagent (Fig. 4D) and by determination of the acetyl bromide lignin level (Fig. 4E). Mäule staining (Fig. 4D) and thioacidolysis (Fig. 4F) revealed a dramatic decrease in S monomers in the mutant compared with Ler, leading to a reduction in S/G ratio from 0.24 to 0.05 (Fig. 4F).

Expression of Medicago CCR1 in the irx4 background led to complete recovery of visible phenotype (Fig. 4B), extractable CCR activity toward feruloyl CoA, total lignin production, and S/G ratio (Fig. 4 D–F). However, CCR activity toward caffeoyl CoA increased by 3.6-fold relative to that in irx4 but attained only 48% of that in Ler (Fig. 4C).

Medicago CCR2 partially complemented the irx4 phenotype; leaf shape and color were more like these of wild-type Ler, but plant growth, although increased, did not reach that of Ler (Fig. 4B). Expression of CCR2 led to a large increase in extractable CCR activity toward caffeoyl CoA, by 17-fold compared with that in irx4 and 2.5 times that in Ler (Fig. 4C), but activity toward feruloyl CoA increased only to about 30% of that in Ler. Total lignin, G and S monomer levels, and S/G ratio were partially restored upon expression of CCR2 in irx4 (Fig. 4 D–F).

Cross-Talk Between the CCR1 and CCR2 Pathways.

The substrate preferences of CCR1 and CCR2 suggest that they probably operate in parallel and potentially competing pathways to coniferaldehyde from caffeoyl CoA, through methylation followed by reduction (the CCoAOMT/CCR1 route) or reduction followed by methylation (the CCR2/COMT route) (Fig. 5). Both CCR1 and CCoAOMT transcript levels were increased in the two ccr2 mutant lines (Fig. S3A), and CCoAOMT protein level (Fig. S3B) and extractable enzymatic activity (Fig. S3C) likewise were increased, suggesting cross-talk between the two potential routes to coniferaldehyde.

Fig. 5.

Fig. 5.

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Alternative routes to coniferaldehyde in Medicago. CCR2 is substrate-inhibited by high concentrations of feruloyl CoA. It is not clear whether the two pools of coniferaldehyde are functionally identical. The enzymes are shown as connecting spheres to depict the possibility of metabolic channeling.

To determine whether such cross-talk might be reciprocal, we screened the M. truncatula Tnt1 mutant population for CCoAOMT knockout lines. Two independent lines had Tnt1 insertions at position 278 in the second intron (NF5183, ccoaomt-1) and at position 1,032 (NF5347, ccoaomt-2) in the fifth exon of the CCoAOMT genomic sequence (Fig. S4A). These lines exhibited strong reduction or complete loss of CCoAOMT transcripts (Fig. S4B) and CCoAOMT protein (Fig. S4C). Homozygous CCoAOMT Tnt1 insertion lines were of somewhat smaller stature than heterozygotes (Fig. S4D) and possessed elevated CCR2 transcript levels (Fig. S3D). Extractable CCR2 activity (against caffeoyl CoA), likewise, was increased in the ccoaomt mutants (Fig. S3F), but activity against feruloyl CoA was much less affected. The ccoaomt mutants also showed increased levels of COMT transcripts and immunodetectable COMT protein (Fig. S3 D and E).

Co–Down-Regulation of CCoAOMT and COMT.

Generation of a ccr1:ccr2 double mutant is problematical because of the already severe phenotype of the ccr1 mutant. Loss of function of CCoAOMT in either alfalfa (5, 9) or M. truncatula has much less effect on plant stature and lignin levels than down-regulation of CCR1, and COMT down-regulation does not affect stature in alfalfa. We therefore addressed the redundancy of the potentially parallel pathways by co–down-regulating CCoAOMT and COMT, making use of existing alfalfa lines in which each gene was individually down-regulated by RNAi (5). Crossing these lines led to identification of progeny with levels of both enzymes down-regulated (Table S3). These plants were dwarf with severely delayed development (Fig. S5A). Acetyl bromide lignin levels were reduced in all COMT and CCoAOMT single-RNAi progeny when compared with the nulls, with the exception of CCoAOMT line 5, and were reduced further in some, but not all, of the double knockdowns (Fig. S5B). The reduction in lignin in the double knockdowns was more apparent when considered in terms of total thioacidolysis yield (Fig. S5C).

Discussion

Substrate preference and kinetic properties of Medicago CCR2 differ from those of previously characterized CCRs. Genetic analysis suggests that CCR2 contributes to constitutive lignin synthesis through the production of caffeyl aldehyde in an alternative pathway, bypassing the requirement for CCoAOMT (Fig. 5). It also is possible that CCR2 contributes to H-lignin formation, because its expression is higher in young internodes, where H-lignin is first laid down, than in more mature internodes; however, CCR1 is expressed at higher levels than CCR2 in young internodes and also is active with coumaroyl CoA.

Like most characterized CCRs (17, 18, 21), Medicago CCR1 prefers feruloyl and sinapoyl CoAs. In contrast, these compounds strongly inhibit CCR2 activity at concentrations above 50 μM. Caffeoyl CoA and 4-coumaroyl CoA, poor substrates for CCR1, are potential allosteric activators (based on positively cooperative kinetics) and are favored substrates for CCR2. Substrate inhibition has been reported recently for a CCR from switchgrass, but this enzyme appears to be associated with defense rather than constitutive lignification (27), as does the second CCR isoform (CCR2) in Arabidopsis (which is, however, also expressed in vascular tissues) (17).

Down-regulation of CCR through antisense expression dramatically reduces lignin levels in alfalfa (20), poplar (28), and tobacco (15), and knockout mutants of Arabidopsis CCR1 showed stunted growth and delayed development (26, 29, 30). The growth of Medicago CCR1 knockout mutants was so impaired that most did not survive under greenhouse conditions. The difference in the phenotypes between Arabidopsis and Medicago CCR1 knockouts can be explained by the different biochemical properties of the second CCR form in these two species. Arabidopsis CCR2 shows reasonable activity with feruloyl and sinapoyl CoAs (17), and its expression is increased in CCR1 knockout plants (17). In contrast, Medicago CCR2, although active with feruloyl and sinapoyl CoAs at low concentrations, is unable to turn these substrates over at concentrations above 50 μM, and no increase was found in CCR2 expression level in ccr1 knockout mutants. The increased levels of feruloyl CoA as a result of knockout of Medicago CCR1 therefore might be too high to be turned over by CCR2, thus resulting in the severe inhibition of lignin biosynthesis.

A reduced S/G ratio was observed in the noncondensed lignin from the ccr1 mutants, whereas ccr2 mutants showed an increased S/G ratio. The effect of CCR repression on S/G ratio appears to be species-dependent. A higher ratio was observed on antisense repression of CCR in tobacco (15, 31), no change was recorded in poplar (28), and a decreased ratio was found in irx4 Arabidopsis mutants (19). Developmental stage and growth conditions affect the S/G ratio in Arabidopsis ccr1 mutants (19, 26, 30), and the decreased S/G ratio in Medicago ccr1 mutants also might be caused by their delayed development. The increased S/G ratio in Medicago ccr2 mutants resulted primarily from decreased G unit levels, perhaps because CCR2 is expressed mainly in xylem vessels, which have little S lignin.

The relative properties and expression patterns of Medicago CCR1 and CCR2 reported here provide a possible explanation for the observation that transgenic alfalfa with severely reduced CCoAOMT activity still makes normal levels of S lignin (9), a finding inconsistent with the scheme for monolignol biosynthesis shown in Fig. S1 (32, 33). CCR2 is expressed in the same cell types as CCoAOMT (34, 35) and shows positively cooperative kinetics with caffeoyl CoA, with increasingly efficient conversion to caffeyl aldehyde as caffeoyl CoA levels increase. Caffeyl aldehyde is a preferred substrate for 3-O-methylation by Medicago COMT (7). Thus, CCR2 and COMT can act together to perform the 3-O-methylation of monolignol precursors in plants in which the CCoAOMT/CCR1 pathway is not functional. This hypothesis is supported by the reciprocal cross-talk between the two pathways. The possession of two nonredundant CCRs may impart an adaptive significance by enabling the plasticity of monolignol biosynthesis under a range of developmental conditions that place different demands on the utilization of hydroxycinnamate thioesters.

Co–down-regulation of COMT and CCoAOMT leads to very severe growth phenotypes in both alfalfa (this work) and Arabidopsis (25). This result is consistent with COMT acting redundantly with CCoAOMT as a 3-O-methyltransferase (25), because loss of the 5-O-methyltransferase function of COMT does not lead to significant growth inhibition. Arabidopsis ccr1 mutants exhibit a smaller reduction in extractable CCR activity against caffeoyl CoA than against feruloyl CoA, suggesting the existence of an additional enzyme with preference for caffeoyl CoA in Arabidopsis. Thus, the alternative pathway described here was predictable from the previous results of downregulating both COMT and CCoAOMT in Arabidopsis (25) and thus may not be limited to Medicago.

The characterization of Medicago CCR2 provides direct experimental evidence for the operation of a shunt in the monolignol pathway via caffeyl aldehyde, further indicates that the designation of COMT as a 5-hydroxyconiferaldehyde O-methyltransferase that functions only in S lignin biosynthesis in vivo (8, 33) was perhaps premature, and opens up interesting avenues for exploring transcriptional crosstalk in monolignol biosynthesis.

Materials and Methods

Plant Materials and Growth Conditions.

M. truncatula plants were grown in the greenhouse under standard conditions in 3.74 l pots in Metro-mix 350 (Scott) with a photoperiod of 16 h light from 0600 h to 2200 h facilitated by supplementary lighting at 25 °C and 8 h in the dark at 22 °C. Transgenic alfalfa plants were derived from field-grown F1 progeny of COMT and CCoAOMT RNAi lines as described in SI Materials and Methods.

Expression of CCR1 and CCR2 in E. coli.

The coding regions of CCR1 and CCR2 were amplified from the original cDNA clones with introduction of NheI enzyme sites before the ATG start codons, a BamHI site after the CCR1 stop codon, and an XhoI site after the CCR2 stop codon. Primers are listed in Table S2. PCR products were cloned into T-vector (Promega). After confirmation by sequencing, both CCR sequences were subcloned into pET28a vector (Novagen), and the resulting constructs were introduced into E. coli strain BL21 (DE34) (Novagen). The enzymes were expressed and purified as described in SI Materials and Methods.

Enzyme Activity Assay.

Assay of purified CCR proteins and HPLC analysis of products are described in SI Materials and Methods. For kinetic analysis, enzyme velocity curves were analyzed using Sigmaplot 10 software (Systat Software, Inc.).

For measurement of enzyme activities in crude plant extracts, crude extracts were prepared as described (9). Caffeoyl CoA (50 μM) was used as substrate to estimate CCR2 activity, and feruloyl CoA (50 μM) was used for estimation of CCR1 activity. Because the crude extract contains cinnamyl alcohol dehydrogenase, which converts cinnamyl aldehydes to the corresponding alcohols, the cinnamyl alcohols also were measured by HPLC analysis and counted toward the CCR activity. Measurement of CCoAOMT activity was as described (9).

Protein Gel Blot Analysis.

Crude proteins (10 μg) were separated on 4–15% gradient SDS–polyacrylamide gels and were electrotransferred onto nitrocellulose membranes. The membranes were incubated for 2 h in blocking buffer (PBS containing 0.05% Tween 20 [Sigma] and 5% skim milk) and then were incubated for 2 h in blocking buffer with antibodies raised against COMT and CCoAOMT protein. Signals were detected with ECL protein gel blotting detection reagents (Amersham) according to the manufacturer's protocol.

Analysis of Lignin.

The sixth internodes (from the top) of M. truncatula stems were cross-sectioned to 100 μm with a vibratome. UV autofluorescence, phloroglucinol, and Mäule staining were performed as described previously (36). The lignin content of stem material was determined by the acetyl bromide method (37), and lignin content and composition were determined further by thioacidolysis (38) as described previously (5).

Real-Time PCR Analysis.

RNA extraction, cDNA synthesis, and quantitative RT-PCR (qRT-PCR) analysis were performed as described previously (27). The raw threshold cycle (Ct) values were normalized against ubiquitin RNA to obtain ΔCt values, which were used to calculate the difference in expression levels. Expression levels (E) were calculated according to the equation: E = Peff (−ΔCt) where Peff is the primer set efficiency calculated using LinRegPCR (39). The primers used are listed in Table S2.

Identification of Retrotransposon Insertion Lines.

M. truncatula lines harboring transposon insertions in CCR1, CCR2, and CCoAOMT were screened from a population of about 15,000 Tnt1 insertional mutant lines (ecotype R108) (24, 40) by PCR-based reverse screening. Primer sequences are listed in Table S2.

Complementation of the irx4 Mutant of Arabidopsis and Expression of Medicago CCRs in Wild Type.

Gateway cloning of the ORFs of CCR1 and CCR2, mobilization to Agrobacterium tumefaciens, and genetic transformation of Arabidopsis were as described in SI Materials and Methods.

In Situ Hybridization.

In situ hybridization was performed as described in SI Materials and Methods.

Supplementary Material

Supporting Information
supp_107_41_17803__index.html (833B, html)

Acknowledgments

We thank Drs. Xiaofei Cheng and Jiangqi Wen for screening transposon insertion lines, Stacy Allen for qRT-PCR analysis, Junying Ma and Yuhong Tang for assistance with in situ hybridization, and Drs. Luis Escamilla-Trevino and Xiaoqiang Wang for critical reading of the manuscript. The M. truncatula plants used in this work were created through research funded, in part, by National Science Foundation Grant 703285. This work was supported by Grant DE-FG02-06ER64303 from the US Department of Energy Feedstock Genomics program (to R.A.D.) with additional support from Forage Genetics International.

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1012900107/-/DCSupplemental.

References

  • 1.Dixon RA, Chen F, Guo D, Parvathi K. The biosynthesis of monolignols: A “metabolic grid”, or independent pathways to guaiacyl and syringyl units? Phytochemistry. 2001;57:1069–1084. doi: 10.1016/s0031-9422(01)00092-9. [DOI] [PubMed] [Google Scholar]
  • 2.Humphreys JM, Chapple C. Rewriting the lignin roadmap. Curr Opin Plant Biol. 2002;5:224–229. doi: 10.1016/s1369-5266(02)00257-1. [DOI] [PubMed] [Google Scholar]
  • 3.Hoffmann L, et al. Silencing of hydroxycinnamoyl-coenzyme A shikimate/quinate hydroxycinnamoyltransferase affects phenylpropanoid biosynthesis. Plant Cell. 2004;16:1446–1465. doi: 10.1105/tpc.020297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Schoch G, et al. CYP98A3 from Arabidopsis thaliana is a 3′-hydroxylase of phenolic esters, a missing link in the phenylpropanoid pathway. J Biol Chem. 2001;276:36566–36574. doi: 10.1074/jbc.M104047200. [DOI] [PubMed] [Google Scholar]
  • 5.Chen F, et al. Multi-site genetic modulation of monolignol biosynthesis suggests new routes for formation of syringyl lignin and wall-bound ferulic acid in alfalfa (Medicago sativa L.) Plant J. 2006;48:113–124. doi: 10.1111/j.1365-313X.2006.02857.x. [DOI] [PubMed] [Google Scholar]
  • 6.Lee D, Meyer K, Chapple C, Douglas CJ. Antisense suppression of 4-coumarate:coenzyme A ligase activity in Arabidopsis leads to altered lignin subunit composition. Plant Cell. 1997;9:1985–1998. doi: 10.1105/tpc.9.11.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Parvathi K, Chen F, Guo D, Blount JW, Dixon RA. Substrate preferences of O-methyltransferases in alfalfa suggest new pathways for 3-O-methylation of monolignols. Plant J. 2001;25:193–202. doi: 10.1046/j.1365-313x.2001.00956.x. [DOI] [PubMed] [Google Scholar]
  • 8.Osakabe K, et al. Coniferyl aldehyde 5-hydroxylation and methylation direct syringyl lignin biosynthesis in angiosperms. Proc Natl Acad Sci USA. 1999;96:8955–8960. doi: 10.1073/pnas.96.16.8955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Guo D, Chen F, Inoue K, Blount JW, Dixon RA. Downregulation of caffeic acid 3-O-methyltransferase and caffeoyl CoA 3-O-methyltransferase in transgenic alfalfa. impacts on lignin structure and implications for the biosynthesis of G and S lignin. Plant Cell. 2001;13:73–88. doi: 10.1105/tpc.13.1.73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Marita JM, et al. Structural and compositional modifications in lignin of transgenic alfalfa down-regulated in caffeic acid 3-O-methyltransferase and caffeoyl coenzyme A 3-O-methyltransferase. Phytochemistry. 2003;62:53–65. doi: 10.1016/s0031-9422(02)00434-x. [DOI] [PubMed] [Google Scholar]
  • 11.Marita JM, Ralph J, Lapierre C, Jouanin L, Boerjan W. NMR characterization of lignins from transgenic poplars with suppressed caffeic acid O-methyltransferase activity. J Chem Soc Perkin Trans 1. 2001:2939–2945. [Google Scholar]
  • 12.Li X, Weng JK, Chapple C. Improvement of biomass through lignin modification. Plant J. 2008;54:569–581. doi: 10.1111/j.1365-313X.2008.03457.x. [DOI] [PubMed] [Google Scholar]
  • 13.Weng JK, et al. Convergent evolution of syringyl lignin biosynthesis via distinct pathways in the lycophyte Selaginella and flowering plants. Plant Cell. 2010;22:1033–1045. doi: 10.1105/tpc.109.073528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Lacombe E, et al. Cinnamoyl CoA reductase, the first committed enzyme of the lignin branch biosynthetic pathway: Cloning, expression and phylogenetic relationships. Plant J. 1997;11:429–441. doi: 10.1046/j.1365-313x.1997.11030429.x. [DOI] [PubMed] [Google Scholar]
  • 15.Piquemal J, et al. Down-regulation of cinnamoyl-CoA reductase induces significant changes of lignin profiles in transgenic tobacco plants. Plant J. 1998;13:71–83. [Google Scholar]
  • 16.Costa S, Dolan L. Epidermal patterning genes are active during embryogenesis in Arabidopsis. Development. 2003;130:2893–2901. doi: 10.1242/dev.00493. [DOI] [PubMed] [Google Scholar]
  • 17.Lauvergeat V, et al. Two cinnamoyl-CoA reductase (CCR) genes from Arabidopsis thaliana are differentially expressed during development and in response to infection with pathogenic bacteria. Phytochemistry. 2001;57:1187–1195. doi: 10.1016/s0031-9422(01)00053-x. [DOI] [PubMed] [Google Scholar]
  • 18.Li L, et al. Clarification of cinnamoyl co-enzyme A reductase catalysis in monolignol biosynthesis of Aspen. Plant Cell Physiol. 2005;46:1073–1082. doi: 10.1093/pcp/pci120. [DOI] [PubMed] [Google Scholar]
  • 19.Patten AM, et al. Reassessment of effects on lignification and vascular development in the irx4 Arabidopsis mutant. Phytochemistry. 2005;66:2092–2107. doi: 10.1016/j.phytochem.2004.12.016. [DOI] [PubMed] [Google Scholar]
  • 20.Jackson LA, et al. Improving saccharification efficiency of alfalfa stems through modification of the terminal stages of monolignol biosynthesis. BioEnergy Research. 2008;1:180–192. [Google Scholar]
  • 21.Goffner D, et al. Purification and characterization of cinnamoyl coenzyme A:NADP oxidoreductase in Eucalyptus gunnii. Plant Physiol. 1994;106:625–632. doi: 10.1104/pp.106.2.625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Ma Q-H. Characterization of a cinnamoyl-CoA reductase that is associated with stem development in wheat. J Exp Bot. 2007;58:2011–2021. doi: 10.1093/jxb/erm064. [DOI] [PubMed] [Google Scholar]
  • 23.Benedito VA, et al. A gene expression atlas of the model legume Medicago truncatula. Plant J. 2008;55:504–513. doi: 10.1111/j.1365-313X.2008.03519.x. [DOI] [PubMed] [Google Scholar]
  • 24.Tadege M, et al. Large-scale insertional mutagenesis using the Tnt1 retrotransposon in the model legume Medicago truncatula. Plant J. 2008;54:335–347. doi: 10.1111/j.1365-313X.2008.03418.x. [DOI] [PubMed] [Google Scholar]
  • 25.Do CT, et al. Both caffeoyl Coenzyme A 3-O-methyltransferase 1 and caffeic acid O-methyltransferase 1 are involved in redundant functions for lignin, flavonoids and sinapoyl malate biosynthesis in Arabidopsis. Planta. 2007;226:1117–1129. doi: 10.1007/s00425-007-0558-3. [DOI] [PubMed] [Google Scholar]
  • 26.Jones L, Ennos AR, Turner SR. Cloning and characterization of irregular xylem4 (irx4): A severely lignin-deficient mutant of Arabidopsis. Plant J. 2001;26:205–216. doi: 10.1046/j.1365-313x.2001.01021.x. [DOI] [PubMed] [Google Scholar]
  • 27.Escamilla-Treviño LL, et al. Switchgrass (Panicum virgatum) possesses a divergent family of cinnamoyl CoA reductases with distinct biochemical properties. New Phytol. 2010;185:143–155. doi: 10.1111/j.1469-8137.2009.03018.x. [DOI] [PubMed] [Google Scholar]
  • 28.Leplé JC, et al. Downregulation of cinnamoyl-coenzyme A reductase in poplar: Multiple-level phenotyping reveals effects on cell wall polymer metabolism and structure. Plant Cell. 2007;19:3669–3691. doi: 10.1105/tpc.107.054148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Goujon T, et al. Down-regulation of the AtCCR1 gene in Arabidopsis thaliana: Effects on phenotype, lignins and cell wall degradability. Planta. 2003;217:218–228. doi: 10.1007/s00425-003-0987-6. [DOI] [PubMed] [Google Scholar]
  • 30.Mir Derikvand M, et al. Redirection of the phenylpropanoid pathway to feruloyl malate in Arabidopsis mutants deficient for cinnamoyl-CoA reductase 1. Planta. 2008;227:943–956. doi: 10.1007/s00425-007-0669-x. [DOI] [PubMed] [Google Scholar]
  • 31.Chabannes M, et al. Strong decrease in lignin content without significant alteration of plant development is induced by simultaneous down-regulation of cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) in tobacco plants. Plant J. 2001;28:257–270. doi: 10.1046/j.1365-313x.2001.01140.x. [DOI] [PubMed] [Google Scholar]
  • 32.Besseau S, et al. Flavonoid accumulation in Arabidopsis repressed in lignin synthesis affects auxin transport and plant growth. Plant Cell. 2007;19:148–162. doi: 10.1105/tpc.106.044495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Vanholme R, Morreel K, Ralph J, Boerjan W. Lignin engineering. Curr Opin Plant Biol. 2008;11:278–285. doi: 10.1016/j.pbi.2008.03.005. [DOI] [PubMed] [Google Scholar]
  • 34.Chen C, et al. Cell-specific and conditional expression of caffeoyl-coenzyme A-3-O-methyltransferase in poplar. Plant Physiol. 2000;123:853–867. doi: 10.1104/pp.123.3.853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Zhong R, Morrison WH, 3rd, Himmelsbach DS, Poole FL, 2nd, Ye ZH. Essential role of caffeoyl coenzyme A O-methyltransferase in lignin biosynthesis in woody poplar plants. Plant Physiol. 2000;124:563–578. doi: 10.1104/pp.124.2.563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Nakashima J, Chen F, Jackson L, Shadle G, Dixon RA. Multi-site genetic modification of monolignol biosynthesis in alfalfa (Medicago sativa): Effects on lignin composition in specific cell types. New Phytol. 2008;179:738–750. doi: 10.1111/j.1469-8137.2008.02502.x. [DOI] [PubMed] [Google Scholar]
  • 37.Hatfield RD, Grabber J, Ralph J, Brei K. Using the acetyl bromide assay to determine lignin concentrations in herbaceous plants: Some cautionary notes. J Agric Food Chem. 1999;47:628–632. doi: 10.1021/jf9808776. [DOI] [PubMed] [Google Scholar]
  • 38.Lapierre C, Monties B, Rolando C. Thioacidolysis of lignin. Comparison with acidolysis. Journal of Wood Chemistry and Technology. 1985;5:277–292. [Google Scholar]
  • 39.Ramakers C, Ruijter JM, Deprez RH, Moorman AFM. Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett. 2003;339:62–66. doi: 10.1016/s0304-3940(02)01423-4. [DOI] [PubMed] [Google Scholar]
  • 40.Tadege M, Ratet P, Mysore KS. Insertional mutagenesis: A Swiss Army knife for functional genomics of Medicago truncatula. Trends Plant Sci. 2005;10:229–235. doi: 10.1016/j.tplants.2005.03.009. [DOI] [PubMed] [Google Scholar]

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