Impact of the Brown-Midrib - American Chemical Society

Article pubs.acs.org/JAFC

Impact of the Brown-Midrib bm5 Mutation on Maize Lignins Valérie Méchin,†,‡ Aurélia Laluc,†,‡ Frédéric Legée,†,‡ Laurent Cézard,†,‡ Dominique Denoue,§ Yves Barrière,§ and Catherine Lapierre*,†,‡ †

INRA, Institut Jean-Pierre Bourgin (IJPB), UMR1318, Saclay Plant Sciences, Route de St-Cyr, 78000 Versailles, France Agroparistech, Institut Jean-Pierre Bourgin (IJPB), UMR1318, Saclay Plant Sciences, Route de St-Cyr, 78000 Versailles, France § INRA, Unité de Génétique et d’Amélioration des Plantes Fourragères (UGAPF), UR889, Route de Saintes, 86600 Lusignan, France ‡

S Supporting Information *

ABSTRACT: We have investigated the impact of the brown-midrib bm5 mutation on lignins and on p-coumaric acid and ferulic acid ester-linked to maize (Zea mays L.) cell walls. Lignified stalks or plant aerial parts (without ears) collected at grain maturity were studied in three genetic backgrounds. Relative to the control, bm5 mutants displayed lower levels of lignins and of pcoumarate esters but increased levels of ferulate esters. Thioacidolysis revealed that bm5 lignins display an increased frequency of free-phenolic guaiacyl units. More importantly, thioacidolysis provided unusual amounts of 1,2,2-trithioethyl ethylguaiacol, a marker compound diagnostic for the incorporation of free ferulic acid into lignins by bis 8-O-4 cross-coupling. As the resulting acetal bonding pattern is a chemically labile branch point introduced in maize lignins by the bm5 mutation, this alteration is prone to facilitate the delignification pretreatments used in the cellulose-to-ethanol process. KEYWORDS: Zea mays L., brown-midrib lignin, ferulic acid, p-coumaric acid, thioacidolysis

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INTRODUCTION Maize brown-midrib (bm) natural mutants have been investigated since the 1930s.1,2 These mutants display reduced lignin together with higher cell-wall digestibility. Among the six bm1−bm6 maize mutations identified and mapped up to date,2,3 the most extensively studied are bm1 and bm3, which reduce lignin content and affect lignin structure. Thioacidolysis, an analytical degradation method providing specific monomers from p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) conventional phenylpropane units,4 revealed the incorporation of unusual levels of 5-hydroxyguaiacyl (5-OH G) units in bm3 lignins, as early as in 1988.5 Later studies established that such a 5-OH G increase is the hallmark of caffeoyl-O-methyltransferase (COMT) deficiency in angiosperm lignins.1 Likewise, thioacidolysis revealed that bm1 maize lignins contain an unusual level of sinapaldehyde linked at its C8 position, a phenomenon diagnostic of cinnamyl alcohol dehydrogenase (CAD) deficiency in angiosperms.1 More recently, the bm2 mutation has been shown to affect the gene encoding for methylenetetrahydrofolate reductase which is involved in the pathway leading to S-adenosyl methionine, the cofactor methyl donor of lignin-specific methyltransferases.3 This mutation induces a lower lignin level and a higher thioacidolysis or pyrolysis S/G ratio but no incorporation of unusual units in lignins. The spontaneous bm5 mutation has been shown to be non allelic to bm1−bm4 (ref 6), and it has been mapped to maize chromosome 5, near the centromere and in the same bin 5.04 as bm1.7 To date, neither the gene(s) responsible for the bm5 mutation nor the precise impact of this mutation on maize lignification has been elucidated. Lignins detrimentally affect both forage digestibility and fermentable sugar yields during the enzymatic cellulose-toethanol conversion process.8,9 The most extensively studied bm1 and bm3 mutations, which both reduce lignin content and © 2014 American Chemical Society

alter lignin structure, are known to increase the enzymatic degradability of maize cell walls.1,2 With the objective to evaluate whether the bm5 mutation might similarly reduce the recalcitrance of lignified cell walls to enzymatic breakdown, the impact of the bm5 mutation on maize lignins was investigated in the present study.

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MATERIALS AND METHODS Chemical. ACS grade solvents (dichloromethane, dioxane, ethanol, pyridine) were purchased from Carlo Erba Reagents (Val de Reuil, France). Analytical reagent grade anhydrous sodium sulfate, BF3 etherate, ethanethiol, heinecosane, N,Obis(trimethylsilyl)trifluoroacetamide, sodium hydrogenocarbonate, and 96% sulfuric acid solution were purchased from Sigma-Aldrich (St Quentin, France). Plant Material. Isogenic bm5 seeds in the genetic backgrounds of two different inbred lines, namely, U740 (U740 and U740bm5) and 5803H (5803H and 5803Hbm5), were obtained from the Maize Genetics Cooperation Stock Center (MGCSC, USDA/ARS, University of Illinois). Mature stems were collected at the grain maturity stage from greenhouse-grown plants at INRA Lusignan in the summer of 2010, with biological duplicates and five plants collected per line. In addition, the bm5 mutation was introgressed into the early flint F2 INRA inbred line. Among the progeny plants grown in the greenhouse in the summer of 2011, whole plants without ears were collected at the grain maturity stage and divided into two groups: F2 × U740bm5 plants displaying the Received: Revised: Accepted: Published: 5102

February 4, 2014 May 13, 2014 May 13, 2014 May 13, 2014 dx.doi.org/10.1021/jf5019998 | J. Agric. Food Chem. 2014, 62, 5102−5107

Journal of Agricultural and Food Chemistry

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mode with a source at 220 °C, an interface at 280 °C, and a 50 to 650 m/z scanning range. The column used was a VF-1 ms 15 m × 0.25 mm i.d. polydimethylsiloxane capillary column (0.25 μm film thickness) (Agilent Technologies, Les Ulis, France) operated in the temperature program mode (from 45 to 180 °C at +30 °C/min, then 180 to 260 °C at +3 °C/min), with helium carrier gas at a 1.5 mL/min flow rate. The GC-MS determinations of the H, G, and S lignin-derived monomers were carried out on ion chromatograms respectively reconstructed at m/z 239, 269, and 299, as compared to the internal standard hydrocarbon evaluated on the ion chromatogram reconstructed at m/z (57 + 71 + 85).12 In addition, the marker compound diagnostic for ferulic acid incorporation in lignins, referred to as the AG marker,13 was evaluated from ion chromatograms reconstructed at m/z 269. The evaluation of the frequency of free phenolic groups within the H, G, and S units was performed by the same thioacidolysis method applied to samples comprehensively permethylated according to a published procedure.14 Analysis of p-Coumaric Acid and Ferulic Acid EsterLinked to the Cell Walls. p-Coumaric and ferulic acids linked to the cell walls by ester bonds were released by mild alkaline hydrolysis and then analyzed by high-performance liquid chromatography (HPLC) combined with photodiode array (PDA) detection, as previously described.12,15 Analysis of Arabinoxylan-Bound p-Coumaric and Ferulic Acids. The evaluation of p-coumaric and ferulic acids ester-linked to arabinose units was made according to a recently published mild acidolysis method16 adapted from the trifluoroacetic acid hydrolysis developed earlier.17,18 The 5-O-pcoumaroyl-L-arabinofuranose, 1, and 5-O-feruloyl-L-arabinofuranose, 2, compounds (α and β anomer mixture) (Figure 1) were analyzed by GC-MS of their TMS derivatives, as previously described.16

bm5 phenotype (five plants collected) and F2 × U740bm5 plants displaying a normal phenotype (five plants collected). Samples were first roughly chopped, dried at 65 °C (ventilated oven) for 72 h and then ground with a hammer mill to pass through a 1 mm screen for further analyses. The milled samples were subjected to exhaustive extraction with water, then with ethanol in a Soxhlet apparatus. The recovered extractive-free samples were dried (65 °C) and used for the analyses of lignins and of cell-wall-linked ferulic and p-coumaric acids. Dioxan lignin fractions were isolated from extractive-free U740 and U740bm5 stem samples according to a published procedure.10 Determination of Klason Lignin (KL). Extractive-free sample (approximately 300 mg weighed to the nearest 0.1 mg) was transferred to a 25 mL beaker, and a 72% (w/w) H2SO4 solution, which was prepared from concentrated 96% H2SO4 according to a published procedure,11 was added (3 mL) while stirring with a small glass rod. The mixture was allowed to stand for 2 h at 20 ± 3 °C with rod stirring every 30 min. The content of the beaker was carefully transferred to a 250 mL round flask, and additional water was used to rinse the beaker and to dilute the acid to a final concentration of 5%. The solution was refluxed (sand bath) for 3 h and with occasional shaking. The cooled reaction mixture was quantitatively vacuum-filtered through a preweighed 60 mL porosity 1 filtering Pyrex crucible (Fischer Scientific, Illkirch, France) fitted with a type GF/A glass microfiber filter (WhatmanFisher, Illkirch, France). The insoluble residue was washed free of acid with water and dried at 105 °C overnight. The KL content was calculated from the oven-dry weight of the residue. The crucible with residue was put in a muffle furnace at 550 °C overnight and then cooled before weighting to the nearest 0.1 mg. The KL content was corrected for the presence of acidinsoluble ash. All samples were assayed at least in triplicate. Thioacidolysis and Subsequent Gas Chromatography−Mass Spectrometry (GC-MS) Analyses of the Lignin-Derived Monomers. The thioacidolysis reagent was prepared by adding 10 mL of ethanethiol and 2.5 mL of BF3 etherate to a 100 mL volumetric flask containing 20 mL dioxane and then adjusting the final volume to 100 mL with dioxane. For each sample, 10 mg of extractive-free ground material was put into a 30 mL glass tube fitted with a Teflonlined screwcap, together with 7 mL of freshly prepared thioacidolysis reagent and 0.1 mL of internal standard solution (heinecosane C21, 2.5 mg/mL in CH2Cl2). The closed tubes were then heated for 4 h and at 100 °C (oil bath), with occasional gentle shaking. After tube cooling in ice water, 7 mL of 0.2 M NaHCO3 was put into each tube to quench the excess BF3 etherate. Then, 0.1 mL of HCl (6 M) solution was added to ensure that the pH of the reaction mixture was less than 3, before addition of 7 mL of dichloromethane extraction solvent and tube mixing. About half of the lower organic phase was carefully removed with a Pasteur pipet and dried over anhydrous sodium sulfate. The dried organic extract was then concentrated under reduced pressure to a final volume of about 1 mL. An aliquot of this solution (5 μL) was trimethylsilylated (TMS) with 100 μL of N,O-bis(trimethylsilyl)trifluoroacetamide and 10 μL of ACS-grade pyridine for 1 h at room temperature. The TMS sample was injected (1 μL) onto a GCMS Varian 4000 instrument (Varian, Les Ulis, France) fitted with an autosampler, a splitless injector (280 °C), and an ion trap mass spectrometer operating in the electronic impact

Figure 1. 5-O-p-coumaroyl-L-arabinofuranose, 1 (R = H), and 5-Oferuloyl-L-arabinofuranose, 2 (R = OMe), compounds (α and β anomer mixture) released by mild acidolysis of extractive-free maize samples.

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RESULTS AND DISCUSSION In the present work, we studied the cell wall phenolics (lignins and ester-linked p-hydroxycinnamic acids) in three different maize genetic backgrounds in order to reliably ascertain the effect of the bm5 mutation on these components. We thus analyzed mature stems from two inbred lines provided with the bm5 mutation (5803Hbm5 and U740bm5) as compared to the corresponding controls (5803H and U740). In addition, we crossed the U740bm5 isogenic line with the INRA F2 line in 5103

dx.doi.org/10.1021/jf5019998 | J. Agric. Food Chem. 2014, 62, 5102−5107


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order to comparatively analyze F2 × U740bm5 plants with the bm phenotype and F2 × U740bm5 plants with a normal phenotype. Similar to other maize bm mutants,1,2 the bm5 plants analyzed herein displayed the brown-midrib phenotype together with the reduced Klason lignin (KL) (Table 1). In the three genetic backgrounds provided with the bm5 mutation, the KL levels were reduced by 10−20% as compared to the corresponding controls.

Table 2. Analyses of 5-O-p-Coumaroyl-L-Arabinofuranose, 1, and of 5-O- Feruloyl-L-Arabinofuranose, 2, Released by Mild Acidolysis of Control (Ctrl) and bm5 Extractive-Free Maize Samplesa maize samples line 5803Hb Ctrl bm5 line U740b Ctrl bm5 F2 × U740bm5d no bm phenotype bm phenotype

Table 1. Compositional Analysis of Control (Ctrl) and bm5 Extractive-Free Maize Samplesa maize samples line 5803Hc Ctrl bm5 line U740c Ctrl bm5 F2 × U740bm5e no bm phenotype bm phenotype

Klason lignin (wt %)

p-coumaric acidb (mg/g)

ferulic acidb (mg/g)

18.76 ± 0.55 15.11 ± 0.46d

26.19 ± 1.61 19.53 ± 0.62d

5.10 ± 0.22 5.94 ± 0.73

17.59 ± 0.59 15.62 ± 0.32d

29.98 ± 0.80 23.76 ± 1.30d

5.11 ± 0.12 6.91 ± 0.19d

16.32 ± 0.02 14.30 ± 0.07d

16.22 ± 0.23 11.50 ± 0.74d

4.49 ± 0.24 6.37 ± 0.88d

1 (mg/g)

2 (mg/g)

0.10 ± 0.01 0.16 ± 0.04

4.39 ± 0.20 4.62 ± 0.34

0.27 ± 0.03 0.32 ± 0.05

6.64 ± 0.12 7.72 ± 0.10c

0.35 ± 0.06 0.48 ± 0.07

3.70 ± 0.20 4.72 ± 0.41c

Data are means ± SD (n = 3). bStems of greenhouse-grown plants collected at the grain maturity stage. cSignificantly different from control at p < 0.05 (Anova test). dGreenhouse-grown plants collected without ears and at the grain maturity stage. a

hydrolysis failed to provide such quantitation.19,22 Taken together, these results suggest that the bm5 mutation affects the content of p-coumarate esters linked to the cell walls by reducing both lignin level and lignin p-coumaroylation. According to literature,23,24 both reductions could favorably affect the degradability of maize cell walls. As compared to control levels, mild alkaline hydrolysis released more ferulic acid from U740bm5 and F2 × U740bm5 samples provided with the bm5 mutation (Table 1). Mild acidolysis consistently released more 5-O-feruloyl-L-arabinofuranose from the same bm5 samples and relative to the controls (Table 2). It has been shown that measurable ferulate is reduced by lignification1,25,26 as ferulate cross-links lignins to arabinoxylans not only at its O-4 position27 but also in other bonding modes that resist alkaline hydrolysis.25 Accordingly, the higher level of measurable ferulate esters observed for two bm5 samples could originate from a higher ferulic acid incorporation into their arabinoxylans and/or from their reduced lignin content. The analysis of cell wall polysaccharides in control and bm5 samples did not reveal any substantial change of the arabinose-to-xylose ratio, which suggests that the arabinoxylan substitution degree is not affected by the bm5 mutation. Unusually High Incorporation of Free Ferulic Acid in bm5 Lignins as Revealed by Thioacidolysis. We then evaluated the impact of the bm5 mutation on lignin structure by thioacidolysis. This method provides lignin-specific H, G, and S thioethylated monomers without any interference from ferulate and p-coumarate esters. In addition, the identification of minor, but diagnostic, unusual thioacidolysis monomers has provided major clues to the identification of lignin structural alterations induced by the mutation target gene.5,28−30 On this basis, we performed an in-depth investigation of lignin-derived compounds released by thioacidolysis of extractive-free maize samples provided with the bm5 mutation and as compared to control samples. When calculated in μmol/gram of Klason lignin, the total yield of thioacidolysis monomers is a reflection of the frequency of lignin units only involved in 8-O-4 ether bonds.4 This frequency is reduced by the bm5 mutation as shown by lower thioacidolysis yield relative to the corresponding controls (Table 3). In addition, the relative percentage of H, G, and S thioacidolysis monomers was found to be strongly altered by

Data are means ± SD (n = 3 to 6). bReleased by mild alkaline hydrolysis. cStems of greenhouse-grown plants collected at the grain maturity stage. dSignificantly different from control at p < 0.01 (Anova test). eGreenhouse-grown plants collected without ears and at the grain maturity stage. a

Effect of the bm5 Mutation on Levels of p-Coumarate Esters and Ferulate Esters in Lignified Cell Walls. The bm5 mutation was found to significantly reduce the level of pcoumarate esters in the three bm5 samples analyzed, which were decreased by 20−30% relative to the control values (Table 1). A reduced level of p-coumarate esters has also been found in bm1 and bm3 stems, whereas the level of p-coumarate esters is not affected by the bm2 and bm4 mutation.1,19 The accretion of p-coumaric acid in maize cell walls has long been reported to parallel lignification because most maize p-coumarate esters are incorporated into lignins.20,21 However, the fact that pcoumarate reduction by 20−30% exceeds the 10−20% reduction in lignin in bm5 samples (Table 1) suggests that lignin p-coumaroylation is also affected by the bm5 mutation. This hypothesis was confirmed by the analysis of purified dioxan lignin fractions isolated by acidolysis in refluxing dioxane/H2O (9/1, v/v) containing 0.2 mol/L HCl for 30 min under nitrogen, a method which essentially preserves the pcoumarate esters linked to lignins. Although dioxan lignin fractions were recovered with a similarly high isolation yield from bm5 and control extractive-free U740 stems (60% of the KL lignin level), the bm5 dioxan lignin fraction contained about 10% less p-coumaric acid than the control dioxane lignin. pCoumaric acid released by duplicate mild alkaline hydrolyses was 127.1 ± 1.5 mg/g from U740bm5 dioxan lignin versus 140.9 ± 2.4 from U740 dioxan lignin. By contrast, the pcoumaroylation of maize arabinoxylans was not reduced by the bm5 mutation as revealed by similar levels of 5-O-p-coumaroylL-arabinofuranose released by mild acidolysis of control and bm5 samples (Table 2). It is noteworthy that the mild acidolysis method, run according to a recently published protocol,16 provided the evidence that a weak but measurable amount of p-coumaric acid acylates maize arabinoxylans, whereas previous studies based on trifluoroacetic acid 5104



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Table 3. Thioacidolysis Monomers Released from Extractive-Free Control and bm5 Maize Samplesa H, G, and S main monomers (yield and % molar) maize samples line 5803Hc Ctrl bm5 line U740c Ctrl bm5 F2 × U740bm5e no bm phenotype bm phenotype

(H + G + S) yield (μmol/g lignin)b

%H

%G

%S

AG yield (μmol/g lignin)b

882 ± 85 699 ± 31d

1.5 ± 0.4 4.6 ± 0.3d

43.8 ± 1.4 19.2 ± 0.7d

54.7 ± 1.3 76.2 ± 0.7d

0.25 ± 0.02 4.80 ± 0.11d

813 ± 14 586 ± 12d

1.7 ± 0.1 3.6 ± 0.2d

38.7 ± 1.1 21.0 ± 1.0d

59.6 ± 1.1 75.4 ± 1.2d

0.70 ± 0.15 3.81 ± 0.79d

602 ± 8 485 ± 16d

2.0 ± 0.3 4.7 ± 0.2d

49.8 ± 1.4 27.6 ± 0.1d

48.2 ± 1.7 67.7 ± 0.3d

0.47 ± 0.05 3.77 ± 0.31d

Data are means ± SD (n = 3). bYield expressed in μmoles per gram of Klason lignin. cStems of greenhouse-grown plants collected at the grain maturity stage. dSignificantly different from control at p < 0.05 (Anova test). eGreenhouse-grown plants collected without ears and at the grain maturity stage. a

Figure 2. Partial GC-MS chromatograms reconstructed at m/z 269 of thioacidolysis monomers released from lignified cell walls of the following: (A) 5803H; (B) 5803Hbm5; (C) U740; (D) U740bm5; (E) F2 × U740bm5 without bm phenotype; and (F) U740 × bm5 with bm phenotype. MG: main guaiacyl monomers; AG: marker for ferulic acid incorporation in lignins; * TMS derivatives of G-CHSEt-CHSET-COOH (G = guaiacyl ring), two diastereoisomers produced from thioacidolysis of ferulate esters.

Figure 3. Mass spectrum of AG TMS derivative (M+• not visible, 389 (M+•-Me), 342 (M+•-EtSH), 282 (G-(CSEt)2 with G = TMS guaiacyl ring), 269 (base peak, G-CHSEt+), 135 (CH(SEt)2+), 75 (CH2SEt+), 73 (TMS+).

the bm5 mutation, with a lower level of G monomers and a higher one of S or H monomers (Table 3). Beside these changes in conventional lignin-derived thioacidolysis monomers, we looked for the occurrence of unusual minor monomers. Not unexpectedly, there were neither increased 5OH G monomers nor increased p-hydroxycinnamaldehydederived compounds, corroborating the fact that bm5 mutation does not affect the genes encoding the lignin-specific COMT or CAD enzymes. By contrast, 1,2,2-trithioethyl ethylguaiacol, the thioacidolysis AG molecular marker reported to be diagnostic of the incorporation of free ferulic acid in the lignins from various cinnamoyl CoA reductase (CCR)-deficient angiosperms,13,30,31 was systematically observed in noticeable amounts for the three genotypes provided with the bm5 mutation while detectable as a trace component on the GC-MS traces of control samples (Figure 2). This component could be unambiguously identified from the mass spectrum of its TMS derivative (Figure 3). It was released from samples provided with the bm5 mutation, with a yield accounting for 0.5−1% of the yield of the main (H + G + S) monomers, a level 5- to 20-fold higher than that from control samples (Table 3). Such an increase indicates that free ferulic acid has been incorporated into lignins at its 8 position and by bis 8-O-4 cross-coupling that induces the loss of its

carboxylic group and the formation of an unusual acetal interunit bond.9,13 This cross-coupling mode drastically contrasts with the more conventional cross-couplings of grass ferulate esters and lignin units involving mainly the ferulate O-4 position.32 When subjected to thioacidolysis, ferulate esters with free or etherified O-4 positions only provide free ferulic acid together with its thioethylated addition products, but no AG.13 By contrast, AG was released from CCR-deficient poplar, Arabidopsis or tobacco plants in 5- to 50-fold higher yield than from corresponding controls, a phenomenon diagnostic of ferulic acid incorporation into lignins.13 From the three maize genotypes under examination that are provided with the bm5 mutation, AG was recovered with similarly increased yields as compared to the control level. Higher Frequency of Free-Phenolic G Units in the Lignins of Maize Provided with the bm5 Mutation. In addition to conventional thioacidolysis, we performed thioacidolysis following an exhaustive cell wall methylation.4,14,33 In these conditions, thioacidolysis monomers methylated at C4 (HOMe, GOMe, and SOMe) originate from originally freephenolic and methylatable H, G, or S parent structures, whereas monomers trimethylsilylated at C4 (HOTMS, GOTMS, and SOTMS) originate from originally etherified H, G, and S parent 5105



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Table 4. Percentage of Free Phenolic Groups in 8-O-4 Linked H, G, and S Lignin Units, as Determined by Thioacidolysis of Permethylated Samplesa maize samples

HOMe/(HOMe + HOTMS)b(%)

GOMe/(GOMe + GOTMS)b (%)

SOMe/(SOMe + SOTMS)b (%)

82.5 ± 0.3 90.1 ± 0.3d

53.1 ± 0.3 59.6 ± 1.2d

8.3 ± 0.3 10.7 ± 0.3d

85.4 ± 2.7 87.8 ± 3.0

55.1 ± 1.6 62.1 ± 1.4d

8.0 ± 0.3 9.8 ± 0.2d

87.3 ± 0.3 90.2 ± 0.1d

48.1 ± 0.4 63.7 ± 0.5d

5.7 ± 0.4 8.3 ± 0.3d

c

line 5803H Ctrl bm5 line U740c Ctrl bm5 F2 × U740bm5e no bm phenotype bm phenotype

Data are means ± SD (n = 2). bMonomers methylated at C4 (HOMe, GOMe, and SOMe) originate from free-phenolic parent structures, whereas monomers trimethylsilylated at C4 (HOTMS, GOTMS, and SOTMS) originate from etherified parent structures. cStems of greenhouse-grown plants collected at the grain maturity stage. dSignificantly different from control at p < 0.01 (Anova test). eGreenhouse-grown plants collected without ears and at the grain maturity stage. a

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structures. As shown in Table 4 and in agreement with previously published results,4,33 the 8-O-4 linked H units were essentially free-phenolic units (i.e., 80−90% of H monomers were methylated at C4), whereas S units were prominently internal units etherified at C4 (only 6−8% could be methylated). G units displayed an intermediate behavior and, more importantly, the percentage of free-phenolic G units was substantially increased in the samples provided with the bm5 mutation. As already reported for maize samples,4 the percentage of free-phenolic G units among the 8-O-4 linked G units was in the 50−55% range for the controls, whereas this percentage was in the 60−64% range for the samples with the bm5 mutation (Table 4). The same trend was observed for S units (Table 4). An increased frequency of free-phenolic G units relative to G units involved in 8-O-4 bonds has been already shown in the lignins of various CAD-deficient4 and CCR-deficient plants.13,30 Not unexpectedly, the thioacidolysis AG molecular marker for ferulic acid incorporation into lignins was observed both as C4-OMe and C4-OTMS derivatives on the GC-MS traces of the TMS thioacidolysis reaction mixture obtained from permethylated samples provided with the bm5 mutation. Introduction of Readily Cleavable Acetal Linkages into the Lignin Backbone by the bm5 Natural Mutation. In addition to the moderately reduced lignin content, the bm5 mutation induces substantial alterations in the structure of the lignins from the three maize bm5 genotypes analyzed herein. The most remarkable alteration, which is common to bm5 maize lignins and to the lignins from CCR-deficient plants,13,30,31 is the increased frequency of bis 8-O-4 acetal linkages in the lignin polymers as revealed by the higher yield of the thioacidolysis-derived AG marker. According to recent studies, these acetal bonds are produced by the incorporation of free ferulic acid into the lignin polymers through readily cleavable acetal linkages.9,13 In recent years, research efforts have focused on redesigning lignin structure to make lignins more amenable to the chemical delignification pretreatments which are necessary for cost-effective production of cellulosic ethanol.8,9,34 Such a structural redesigning can not only rely on elegant transgenic strategies as recently reported35 but can also profit from spontaneous mutations like the bm5 maize mutation.

ASSOCIATED CONTENT

* Supporting Information S

GC-MS traces of 5-O-p-coumaroyl-L-arabinofuranose, 1, and of 5-O-feruloyl- L-arabinofuranose, 2, isomers released by acidolysis of extractive-free F2 × U740bm5 sample (α and β anomers analyzed as their TMS derivatives), and corresponding mass spectra (electronic impact 70 eV). GC-MS traces of thioacidolysis monomers from U740bm5 and from permethylated U740bm5 extractive-free samples. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: (33) (0)1-3083-30-99. Tel.: (33)-(0)1-30-83-32-13. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We very sincerely thank Dr. Paul Scott (Iowa State University), Dr. Thomas Lübberstedt (Iowa State University), and Dr. Marty Sachs (MGCSC, University of Illinois) for kindly providing the bm5 seeds in the U740 and 5803H genetic backgrounds. We thank Richard Chazal for performing the analysis of the composition of cell wall polysaccharides. This research was partially funded by the ANR Biomass for the F u tur e p r o g r a m (B F F 2 0 1 2−2019 ; h ttp://www. biomassforthefuture.org/en/) and by the ANR MAGIC 2008−2012 program for financial support to A.L.

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ABBREVIATIONS USED CAD, cinnamyl alcohol dehydrogenase; COMT, caffeoyl-Omethyltransferase; CCR, cinnamoyl CoA reductase; G, guaiacyl; H, p-hydroxyphenyl; KL, Klason lignin; 5-OH G, 5hydroxyguaiacyl; S, syringyl; TMS, trimethylsilylated

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REFERENCES

(1) Barrière, Y.; Ralph, J.; Méchin, V.; Guillaumie, S.; Grabber, J. H.; Argillier, O.; Chabbert, B.; Lapierre, C. Genetic and molecular basis of grass cell wall biosynthesis and degradability. II. Lessons from brownmidrib mutants. C. R. Biol. 2004, 327, 847−860. (2) Sattler, S. E.; Funnell-Harris, D. L.; Pedersen, J. F. Brown midrib mutations and their importance to the utilization of maize, sorghum, and pearl millet lignocellulosic tissues. Plant Sci. 2010, 178, 229−238.

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(22) Hatfield, R. D.; Chaptman, A. K. Comparing corn types for differences in cell wall characteristics and p-coumaroylation of lignin. J. Agric. Food Chem. 2009, 57, 4243−4249. (23) Barrière, Y.; Thomas, J.; Denoue, D. QTL mapping for lignin content, lignin monomeric composition, p-hydroxycinnamate content, and cell wall digestibility in the maize recombinant inbred line progeny F838 × F286. Plant Sci. 2008, 175, 585−595. (24) Zhang, Y.; Culhaoglu, T.; Pollet, B.; Melin, C.; Denoue, D.; Barrière, Y.; Baumberger, S.; Méchin, V. Impact of lignin structure and cell wall reticulation on maize cell wall degradability. J. Agric. Food Chem. 2011, 59, 10129−10135. (25) Grabber, J. H.; Hatfield, R. D.; Ralph, J.; Zon, J.; Amrhein, N. Ferulate cross-linking in cell-walls isolated from maize cellsuspensions. Phytochemistry 1995, 40, 1077−1082. (26) Grabber, J. H.; Ralph, J.; Lapierre, C.; Barrière, Y. Genetic and molecular basis of grass cell-wall degradability. I. Lignin−cell wall matrix interactions. C. R. Biol. 2004, 327, 455−465. (27) Jacquet, G.; Pollet, B.; Lapierre, C. New ether-linked ferulic acid-coniferyl alcohol dimers identified in grass straws. J. Agric. Food Chem. 1995, 43, 2746−2751. (28) Jouanin, L.; Goujon, T.; de Nadai, V.; Martin, M. T.; Mila, I.; Vallet, C.; Pollet, B.; Yoshinaga, A.; Chabbert, B.; Petit-Conil, M.; Lapierre, C. Lignification in transgenic poplars with extremely reduced caffeic acid O-methyltransferase activity. Plant Physiol. 2000, 123, 1363−1373. (29) Kim, H.; Ralph, J.; Lu, F. C.; Pilate, G.; Leplé, J.-C.; Pollet, B.; Lapierre, C. Identification of the structure and origin of thioacidolysis marker compounds for cinnamyl alcohol dehydrogenase deficiency in angiosperms. J. Biol. Chem. 2002, 277, 47412−47419. (30) Mir Derikvand, M.; Sierra, J. B.; Ruel, K.; Pollet, B.; Do, C.-T.; Thévenin, J.; Buffard, D.; Jouanin, L.; Lapierre, C. Redirection of the phenylpropanoid pathway to feruloyl malate in Arabidopsis mutants deficient for cinnamoyl-CoA reductase 1. Planta 2008, 227, 943−956. (31) Leplé, J. C.; Dauwe, R.; Morreel, K.; Storme, V.; Lapierre, C.; Pollet, B.; Naumann, A.; Kang, K. Y.; Kim, H.; Ruel, K.; Lefebvre, A.; Joseleau, J. P.; Grima-Pettenati, J.; De Rycke, R.; Andersson-Gunneras, S.; Erban, A.; Fehrle, I.; Petit-Conil, M.; Kopka, J.; Polle, A.; Messens, E.; Sundberg, B.; Mansfield, S. D.; Ralph, J.; Pilate, G.; Boerjan, W. 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. (32) Grabber, J. H.; Ralph, J.; Hatfield, R. D. Model studies of ferulate-coniferyl alcohol cross-product formation in primary maize walls: Implications for lignification in grasses. J. Agric. Food Chem. 2002, 50, 6008−6016. (33) Lapierre, C.; Monties, B.; Rolando, C. Thioacidolyses of diazomethane-methylated pine compression wood and wheat straw in situ lignins. Holzforschung 1988, 42, 409−411. (34) Fu, C.; Mielenz, J. R.; Xiao, X.; Ge, Y.; Hamilton, C. Y.; Rodriguez, M.; Chen, F.; Foston, M.; Ragauskas, A.; Bouton, J.; Dixon, R. A.; Wang, Z.-Y. Genetic manipulation of lignin reduces recalcitrance and improves ethanol production from switchgrass. Proc. Natl. Acad. Sci. USA 2011, 108, 3803−3808. (35) Wilkerson, C. G.; Mansfield, S. D.; Lu, F.; Withers, S.; Park, J.Y.; Karlen, S. D.; Gonzales-Vigil, E.; Padmakshan, D.; Unda, F.; Rencoret, J.; Ralph, J. Monolignol ferulate transferase introduces chemically labile linkages into the lignin backbone. Science 2014, 344, 90−93.

(3) Tang, H. M.; Liu, S.; Hill-Skinner, S.; Wu, W.; Reed, D.; Yeh, C.T.; Nettleton, D.; Schnable, P. S. The maize brown midrib2 (bm2) gene encodes a methylenetetrahydrofolate reductase that contributes to lignin accumulation. Plant J. 2014, 77, 3806392. (4) Lapierre, C. Determining lignin structure by chemical degradations. In Lignin and lignansAdvances in Chemistry; Heitner, C., Dimmel, D., Schmidt, J. A., Eds.; CRC Press, Taylor & Francis Group: Boca Raton, Florida, 2010; pp 11−48. (5) Lapierre, C.; Tollier, M. T.; Monties, B. Occurrence of additional monomeric units in the lignins from internodes of a brown-midrib mutant of maize bm3. C. R. Acad. Sci. 1988, 307, 723−728. (6) Ali, F.; Scott, P.; Bakht, J.; Chen, Y.; Lübberstedt, T. Identification of novel brown midrib genes in maize by tests of allelism. Plant Breed. 2010, 129, 724−726. (7) Maize genetics and genomics database. URL (http://www. maizegdb.org/) (Accessed: 24 March 2014). (8) Vanholme, R.; Morreel, K.; Ralph, J.; Boerjan, W. Lignin engineering. Curr. Opin. Plant Biol. 2008, 11, 278−285. (9) Vanholme, R.; Morreel, K.; Darrah, C.; Oyarce, P.; Grabber, J. H.; Ralph, J.; Boerjan, W. Metabolic engineering of novel lignin in biomass crops. New Phytol. 2012, 196, 978−1000. (10) Hibino, T.; Shibata, D.; Ito, T.; Tsuchiya, D.; Higuchi, T.; Pollet, B.; Lapierre, C. Chemical properties of lignin from Aralia cordata. Phytochemistry 1994, 37, 445−448. (11) Dence, C. W. The determination of lignin. In Methods in Lignin Chemistry; Lin, S. Y., Dence, C. W., Eds.; Springer-Verlag: Berlin, Germany, 1992; pp 33−61. (12) Bouvier d’Yvoire, M.; Bouchabke-Coussa, O.; Voorend, W.; Antelme, S.; Cézard, L.; Legée, F.; Lebris, P.; Legay, S.; Whitehead, C.; McQueen-Mason, S. J.; Gomez, L. D.; Jouanin, L.; Lapierre, C.; Sibout, R. Disrupting the cinnamyl alcohol dehydrogenase 1 gene (BdCAD1) leads to altered lignification and improved saccharification in Brachypodium distachyon. Plant J. 2013, 73, 496−508. (13) Ralph, J.; Kim, H.; Lu, F.; Grabber, J. H.; Leplé, J.-C.; BerrioSierra, J.; Mir Derikvand, M.; Jouanin, L.; Boerjan, W.; Lapierre, C. Identification of the structure and origin of a thioacidolysis marker compound for ferulic acid incorporation into angiosperm lignins (and an indicator for cinnamoyl CoA reductase deficiency). Plant J. 2008, 53, 368−379. (14) Pitre, F. E.; Pollet, B.; Lafarguette, F.; Cooke, J. E. K.; MacKay, J. J.; Lapierre, C. Effects of increased nitrogen supply on the lignification of poplar wood. J. Agric. Food Chem. 2007, 55, 10306− 10314. (15) Culhaoglu, T.; Zheng, D.; Méchin, V.; Baumberger, S. Adaptation of the Carrez procedure for the purification of ferulic and p-coumaric acids released from lignocellulosic biomass prior to LC/MS analysis. J. Chromatogr. B 2011, 879, 3017−3022. (16) Petrick, D. L.; Karlen, S. D.; Cass, C. L.; Padmakshan, D.; Lu, F.; Liu, S.; Le Bris, P.; Antelme, S.; Santoro, N.; Wilkerson, C. G.; Sibout, R.; Lapierre, C.; Ralph, J.; Sedbrook, J. C. p-Coumaroyl-CoA:monolignol transferase (PMT) acts specifically in the lignin biosynthetic pathway in Brachypodium distachyon. Plant J. 2014, 77, 713−726. (17) Brown, J. A.; Fry, S. C. The preparation and susceptibility to hydrolysis of novel O-galacturonoyl derivatives of carbohydrates. Carbohydr. Res. 1993, 240, 95−106. (18) Saulnier, L.; Vigouroux, J.; Thibault, J. F. Isolation and partial characterization of feruloylated oligosaccharides from maize bran. Carbohydr. Res. 1995, 272, 241−253. (19) Marita, J. M.; Vermerris, W.; Ralph, J.; Hatfield, R. D. Variations in the cell wall composition of maize brown midrib mutants. J. Agric. Food Chem. 2003, 51, 1313−1321. (20) Ralph, J.; Hatfield, R. D.; Quideau, S.; Helm, R. F.; Grabber, J. H.; Jung, H. J. G. Pathway of p-coumaric acid incorporation into maize lignin as revealed by NMR. J. Am. Chem. Soc. 1994, 116, 9448−9456. (21) Hatfield, R. D.; Marita, J. M.; Frost, K. Characterization of pcoumarate accumulation, p-coumaroyl transferase, and cell wall changes during the development of corn stems. J. Sci. Food Agric. 2008, 88, 2529−2537. 5107


Impact of the Brown-Midrib - American Chemical Society

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