LIGNIN-CARBOHYDRATE LINKAGES, LIGNIN, AND THE RELATIONSHIP WITH FIBER DIGESTIBILITY

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1 LIGNIN-CARBOHYDRATE LINKAGES, LIGNIN, AND THE RELATIONSHIP WITH FIBER DIGESTIBILITY H. M. Dann, R. J. Grant, M. E. Van Amburgh and P. J. Van Soest William H. Miner Agricultural Research Institute and Department of Animal Science Cornell University INTRODUCTION Neutral detergent fiber (NDF) digestibility is an important input for CNCPS, CPM- Dairy, and other nutritional models for predicting energy content of forages. Direct measurement of NDF digestibility by commercial laboratories in the US more than doubled from 2002 to 2004 (data from Dairyland Laboratories, Inc., 2005). When NDF digestion is not measured directly, the chemical measure that is most often used to predict NDF digestibility is lignin. It is well recognized that lignin content is a key intrinsic factor that limits cell wall digestibility in forages. Additionally, Jung and Allen (1995) suggested that cross-linkages between lignin/cell wall polysaccharides and phenolic acids (p-coumaric and ferulic acids) might be a prerequisite for lignin to exert its inhibitory effect on cell wall digestibility. Two common phenolic acids, p-coumaric and ferulic acids, are ester- and ether-linked to arabinoxylans (hemicellullose) in grasses. The phenolic acid cross-linkages decrease the enzymatic degradation of forage cell walls by ruminal microbes. It appears that phenolic acids are one of the factors most inhibitory to digestibility of cell wall polysaccharides (Yu et al., 2005), and numerous studies over the years have shown that phenolic acids are in fact negatively correlated with forage digestibility (Hartley, 1972; Burritt et al., 1984; Méchin et al., 2000; Casler and Jung, 2006). Measurement of lignin has remained problematic and various lignin methods result in different values for the same forage sample. Consequently, the true nutritional implications of lignification on plant cell wall digestion are debatable and influenced by the method used to measure lignin. Currently, the two most common methods for measurement of lignin are Klason lignin (KL) and acid detergent lignin (ADL). Because the KL procedure recovers more phenolic compounds from the plant cell wall than ADL, it results in a larger lignin concentration. Implications of these two methods on the relationship between lignin and NDF digestibility are discussed in a later section. Most recently, lignin chemists have proposed that phenolic acid content of forage might be a better predictor of NDF digestibility than lignin content because it would provide information on the crosslinking between lignin and carbohydrates. If proven useful, phenolic acid measurement has the potential to become a routine laboratory analysis. If there is a strong relationship between phenolic acid content of forage and some aspect of NDF digestibility, then a database could be formed that would aid in development of rates and extents of NDF digestion of forages to be used in CNCPS, CPM-Dairy, and other nutritional models.

2 PLANT CELL WALL COMPOSITION AND STRUCTURE Plant cell walls are composed of sugars, such as rhamnose, arabinose, xylose, mannose, galactose, glucose, galacturonic acid, and glucuronic acid, arranged as polysaccharides with various compositions and structures, typically categorized as cellulose, hemicellulose, and pectin, and complexed with hydroxycinnamic acids, lignin, protein, ions, and water (Hatfield et al., 1999; Wang and McAllister, 2002). Hydroxycinnamic acids, such as p-coumaric and ferulic acids, are able to create cross-linkages between cell wall polysaccharides (Figure 1). Figure 1. Linkages (ester and ether cross-links) between carbohydrate and lignin in plant cell walls. Linkages are: 1=direct ester linkage, 2=hydroxycinnamic acid ester, 3=hydroxycinnamic acid ether, 4=ferulic acid bridge, 5=direct ether linkage, 6=dehydrodiferulic acid diester bridge, 7=dehydrodiferulic acid diester-ether bridge. (from Krause et al., 2003) p-coumaric acid is esterified primarily to lignin (Jung and Allen, 1995). Ferulic acid is crosslinked to polysaccharides by ester bonds and to lignin mainly by ether bonds (Jung and Allen, 1995; Yu et al., 2005). Ferulic acid can form dimers and trimers through oxidative coupling between esterified or etherfied residues. As a result of dimer and trimer formation, polysaccharides become extensively cross-linked and incorporated into lignin (Yu et al., 2005). These extensive cross-links are thought to 1) increase mechanical strength of the cell wall, and 2) decrease the nutritional value of the cell wall by reducing nutrient availability by interfering with the attachment of ruminal bacteria to the cell wall by acting as a physical barrier to microbial enzymatic digestion through steric hindrance (Jung and Allen, 1995; Yu et al., 2005). Ruminal microbes have phenolic acid esterases that can break the ester linkages if they are accessible, but microbial cleavage of ether linkages is not known to occur in the anaerobic ruminal environment (Jung and Allen, 1995). Total lignin content of cell walls increases gradually during plant maturation while digestibility of cell walls decreased rapidly. However, the relationship of lignin and digestibility is poor when forages of the same stage of maturity are compared. A negative correlation of lignin content and cell wall digestibility is typically observed across plant maturities, but the relationship is not often observed in plants of similar maturity. The method used to measure lignin also plays an important role in determining the nature of the relationship between lignification and NDF digestibility as will be discussed.

3 Van Amburgh (2004) summarized data from numerous corn hybrids ranging in NDF content from mid-30% to over 50% of dry matter (DM). Despite similar ADL and NDF contents in many instances, the 30-h in vitro NDF digestibility differed by as much as 20 percentage units. To-date, the reason for the large range in NDF digestibility for samples of similar ADL/NDF remains unknown. One potential explanation would be differences in lignin-carbohydrate cross-linking among these corn hybrids. PHENOLIC ACIDS AND THEIR RELATIONSHIP TO DIGESTIBILITY The literature demonstrating a relationship between phenolic acid content, linkage type, and cell wall digestibility has grown over the past several decades. Early research found that Italian ryegrass samples of different maturities had a correlation of 0.98 between cell wall digestibility and the ferulic to p-coumaric acid ratio (Hartley, 1972). The correlation between in vitro dry DM digestibility and p-coumaric acid was -0.86, and the correlation between in vitro DM digestibility and the ratio of p-coumaric to ferulic acid was (Burritt et al., 1984) when reed canary grass, Russian wildrye, and smooth bromegrass of different maturities were evaluated. Similarly, Iiyama and Lam (2001) demonstrated that ferulic acid bridges between lignin and polysaccharides had a negative effect (r = -0.73) on in vitro DM digestibility in approximately 100 varieties of ryegrass. Most recently, Casler and Jung (2006) found that content of NDF, KL, and ferulate ethers were negatively correlated with in vitro NDF digestibility in perennial grasses (reed canarygrass, smooth bromegrass, and cocksfoot). Interestingly, the impact of ferulate cross-linking was more pronounced for potentially digestible NDF (96-h incubations) than the rapidly digestible NDF fraction (24-h incubations). Etherified ferulate had a significant negative effect on 24-h in vitro NDF digestibility of cocksfoot (regression coefficient = -0.31) and reed canarygrass (regression coefficient = -0.14) but not smooth bromegrass. In contrast, etherified ferulate had a significant negative effect on 96-h in vitro NDF digestibility of all three grasses. The regression coefficients were , -0.39, and for smooth bromegrass, cocksfoot, and reed canarygrass, respectively. Méchin et al. (2000) found a large degree of genetic variation for NDF (45-58%), KL (12-20% NDF), phenolic acids (2-28 mg/g NDF), and in vitro NDF digestibility (24-44%) among maize inbred lines. Esterifed p-coumaric acid was negatively correlated to in vitro NDF digestibility (r = -0.78). A multiple regression equation based on esterified p- coumaric acid and lignin composition explained 58% of the in vitro NDF digestibility variation (IVNDFD = esterified p-coumaric acid syringly:guaiacyl unit ratio; Méchin et al., 2000). Interestingly, KL and NDF accounted for only 28% and 25% of the variation of in vitro NDF digestibility, respectively. Jung and Casler (2006) found strong negative correlations of in vitro cell wall digestibility at 24 and 96 h, respectively with p-coumaric acid (r = & -1.00), esterlinked ferulic acid (r = h), ether-linked ferulic acid (r= & -0.94), and KL (r=

4 -0.90 & -0.92) for the fourth elongated internode of maize. Interestingly, when maize internodes from 10 developmental stages were evaluated, the relationship of cell wall digestibility and ferulate cross-linking (measured as ether-linked ferulate) was complex. When the amount of ferulate cross-links of lignin to arabinoxylan increased during internode elongation, cell wall digestibility decreased, but the relationship became positive post-elongation perhaps reflecting dilution of the cross-linked and easily soluble lignin with more highly polymerized core lignin. Most studies conducted have evaluated the relationship of phenolic acids and forage digestibility in vitro or in situ. In contrast, Mandebvu et al. (1999) evaluated Tifton 85 and Coastal bermudagrasses digestibility in vivo using growing beef steers and related the results to cell wall composition. Tifton 85 bermudagrass had a higher content of cell wall (737 vs. 708 g/kg DM) and total neutral sugars (818 vs. 792 g/kg cell wall) and a lower content of ADL (175 vs. 203 g/kg cell wall) and ether-linked ferulic acid (monomers and dimers; 6.9 vs. 8.1 g/kg cell wall) compared to Coastal bermudagrass. The higher content of ADL and ether-linked ferulic acid in Coastal bermudagrass limited total potential extent of digestion of DM (53 vs. 58%) and NDF (58 vs. 66%). We recently completed a study (Dann et al., 2005) to determine 1) the content of p- coumaric and ferulic acids in common forages, and 2) correlations for NDF digestibility and ester-linked, etherlinked, and total p- coumaric and g/100 g NDF Corn silage Ester-linked PCA Ester-linked FA Ether-linked PCA Ether-linked FA BMR Corn Silage Oat Hay ferulic acids. Figure 2. p-coumaric acid (PCA) and ferulic acid (FA) content Digestibility of NDF of forage samples. ranged from less than 20% for poor quality alfalfa hay to 52% for good quality timothy hay. The large range in NDF digestibility for the forages provided a good sample population to evaluate the relationship of phenolic acids with digestibility. As anticipated, the content of ester-linked and ether-linked p-coumaric and ferulic acids varied among forages (Figure 2). Normal corn silage had higher lignin and p- coumaric and ferulic acids than bmr corn silage. Grasses had a higher content of esterlinked and ether-linked p-coumaric and ferulic acids than legumes as shown in previous studies. Ester-linked p-coumaric and ferulic acids were not detected in three of the four alfalfa samples. The content of p-coumaric and ferulic acids in NDF residue of forages remained relatively unchanged during a 96 h in situ fermentation. Oat Straw Alfalfa Hay, 1st Alfalfa Hay, 2nd Alfalfa Hay, 3rd Alfalfa Hay, 4th Timothy Hay, 1st Timothy Hay, 2nd

5 Digestibility of NDF of each forage was determined using in situ analysis at 24 and 48 h (Table 1). In addition, digestibility of NDF was calculated from lignin (NRC, 2001) (Table 1). Correlation analysis was conducted to determine the relationship between p- coumaric and ferulic acids with NDF digestibility (Table 2). There were significant negative correlations between ester-linked phenolic acids and NDF digestibility determined with a 24 h in situ measurement. Total phenolic acids were negatively correlated to NDF digestibility determined with a 24 h in situ measurement. There were no significant correlations with NDF digestibility at 48 h perhaps reflecting a dilution of the effect of the extractable phenolic acids by the more highly polymerized lignin that would be assayed primarily as ADL. Table 1. In situ and calculated NDF digestibility of forages. Alfalfa Hay Timothy Hay Corn bmr Corn Oat Oat 2 nd Item Silage Silage Hay Straw 1 st Cut 2 nd Cut 3 rd Cut 4 th Cut 1 st Cut Cut 1 NDFd IS NDFd IS NDFd Lignin In situ 24 h NDF digestibility (% of NDF). 2 In situ 48 h NDF digestibility (% of NDF). 3 Calculated (NRC, 2001) NDF digestibility (% DM). Table 2. Pearson correlation coefficients for p-coumaric acid (PCA) and ferulic acid (FA) with digestibility and chemical measurements of forages. Ester-linked Ether-linked Total PCA + FA PCA FA PCA + FA PCA FA PCA + FA Item PCA FA 1 NDFd IS * * -0.74* * * 2 NDFd IS NDFd Lignin andf, % DM Lignin, % DM * 1 In situ 24 h NDF digestibility (% NDF). 2 In situ 48 h NDF digestibility (% NDF). 3 Calculated (NRC, 2001) NDF digestibility (% DM). * P < 0.01.

6 We conducted a study (Dann et al., unpublished) to determine in situ NDF digestion kinetics and phenolic acid content of wild-type sorghum stover, bmr-6 sorghum stover, bmr-12 sorghum stover, and stacked bmr-6, bmr-12 sorghum stover (provided to us by J. F. Pedersen, USDA, ARS, NPA Wheat Sorghum and Forage Research, Dep. of Agronomy, Univ. of Nebraska, Lincoln, NE). The bmr-6 mutation results in reduced cinnamyl-alcohol dehydrogenase (CAD) activity (Pillonel et al., 1991), while the bmr-12 and bmr-18 mutations result in reduced caffeic acid O-methyltransferase (COMT) activity (Bout and Vermerris, 2003). Both mutations result in less lignin and presumably a change in lignin composition and the type and extent of cross-linking. Wild-type sorghum stover had a higher content of ADL than bmr-6, bmr-12, or stacked bmr sorghum stover (Table 3). Brown midrib sorghum stover was digested to a greater extent in situ than wild-type sorghum stover (Table 4; Figure 3). Interestingly, the stacked bmr sorghum stover had a greater potential extent of digestion of NDF at 96 h than either bmr-6 or bmr-12 Neutral Detergent Fiber Residue (% of original) Wild-type bmr-6 bmr-12 Stacked bmr Time of Incubation (h) Figure 3. In situ neutral detergent fiber residue of sorghum stover over a 96 h incubation period (lsmeans; SEM = 2.4). Table 3. Chemical composition (DM basis) of the sorghum stover. Sorghum Item Wildtype bmr-6 bmr-12 NE L, Mcal/kg CP, % ADF, % NDF, % ADL % NFC, % Starch, % Sugar, % Crude fat, % Stacked bmr Ash, % (Table 4; Figure 3). Rate of digestion (k d ) in situ was similar among sorghum stover (Table 4). Content of ester p-coumaric acid and ether ferulic acid was greater in wild-type than bmr-6, bmr-12, and stacked bmr sorghum stover over a 96 h in situ incubation period (Figure 4). There was no significant effect of time of fermentation on phenolic acid content. However, there was a significant interaction of sorghum type by time for ester p-courmaric acid (Figure 4).

7 Table 4. In situ neutral detergent fiber digestion kinetics 1 of sorghum stover. Sorghum Item Wild-type bmr-6 bmr-12 Stacked bmr SE Lag, h K 2 d, h PED 3, % 53.1 d 54.7 c 65.3 b 74.1 a 3.1 R Calculated according to the model described by Mertens and Loften (1980). 2 Fractional rate of neutral detergent fiber digestion. 3 Potential extent of digestion of neutral detergent fiber at 96 h of in situ fermentation. a, b, c, d Means within a row with different superscripts differ (P < 0.05). Ester p-coumaric Acid (g/100 g NDF) Wild-type bmr-6 bmr-12 Stacked bmr Ester Ferulic Acid (g/100 g NDF) Wild-type bmr-6 bmr-12 Stacked bmr Time of Incubation (h) Time of Incubation (h) Ether p-coumaric Acid (g/100 g NDF) Wild-type bmr-6 bmr-12 Stacked bmr Ether Ferulic Acid (g/100 g NDF) Wild-type bmr-6 bmr-12 Stacked bmr Time of Incubation (h) Time of Incubation (h) Figure 4. Ester linked p-coumaric acid, ester linked ferulic acid, ether linked p-coumaric acid, and ether linked ferulic acid content of sorghum stover over a 96-h in situ incubation period. DEVELOPING A MODEL OF HOW LIGNIN INHIBITS CELL WALL DIGESTION The prevailing assumption among lignin chemists is that KL measures total plant lignin. In contrast, ADL measures only a part of the core lignin, rather than all of the extractable phenolic compounds in the cell wall. Consequently, the KL procedure yields larger values for lignin than ADL, particularly for grasses. In fact, Van Soest and Van Amburgh (2006, unpublished) re-analyzed lignin and digestibility data from Jung et al. (1997) and demonstrated conclusively that KL comprises two distinct chemical fractions that appear to be equivalent to the ADL and acid detergent dispersible lignin (ADDL) fractions described by Lowry et al. (1994). The difference between KL and ADL is a

8 chemical fraction that comprises less polymerized, polyphenolic compounds that are commonly solubilized into ruminal fluid. Lowry et al. (1994) characterized the properties of an ADDL fraction that is thought to be similar to the L (KL ADL) fraction defined by Van Soest and Van Amburgh (2006). Lowry et al. (1994) emphasized the fact that, regardless of lignin methodology, any analysis of ADF will substantially underestimate grass lignin because a considerable fraction is extracted during acid detergent reflux. Lowry et al. (1994) also pointed out the prevailing definitions of non-core and core lignin. Non-core lignin refers collectively to the monomeric phenolic acids that are esterified directly to arabinoxylans and other phenolic compounds, whereas core lignin has been equated with ADL. The content of non-core lignin has been grossly underestimated due to limitations in lignin methodology that also explain the misinterpretation of grass lignin as being more inhibitory than legume lignin on cell wall digestion. The ADDL fraction is routinely assayed to be greater than the ADL content (as much as 3x greater in some tropical grasses; Lowry et al., 1994). In contrast, legumes have ADDL concentrations much less than ADL concentration. Characteristics of the acid detergent extractable fraction of lignin, and presumably the L fraction as well (Van Soest and Van Amburgh, 2006), that contribute to reduced NDF digestion include (Lowry et al., 1994): 1) It depresses the digestion of the neutral detergent solubles (NDS). The extent of the depression in NDS digestibility is a function of the amount of ADDL. The lignin-carbohydrate complexes comprising the ADDL fraction are resistant to digestion in the digestive tract and appear as part of the NDS fraction in the feces. 2) It influences the rate of NDF digestion, presumably the fast rate of NDF digestion, and hence 3) It influences the amount of NDF that will be digested in the rumen at earlier time points of fermentation such as 24, 30, or 48 h. Most of the difference in apparent digestibility between ADL and ADDL occur within the first 48 h of fermentation (Lowry et al., 1994). 4) Phenolic-carbohydrate fragments (thought to comprise much of the ADDL fraction) could exert a direct inhibitory effect on the activity of fibrolytic bacteria. Wilson and Mertens (1995) describe how phenolic-carbohydrate complexes are more likely to be released than phenolic monomers as a result of cell wall digestion based on research exemplified by Cherney et al. (1992). Complexes isolated by Cherney et al. (1992) and others are substantially larger than phenolic monomers and consequently have a slower diffusion rate and their concentration may increase within the microenvironment of the lumen of a plant cell undergoing active digestion. Wilson

9 and Mertens (1995) calculate, based on diffusion coefficients for molecules of a size similar to the phenolic-carbohydrate fragments, that it would take 36 min to as long as 2.9 h for a complex to traverse a 1.0-mm sclenchyma cell. With this scenario, together with the fact that fibrolytic microbes must remain tightly bound to their substrate during active digestion, the concept of phenolic toxicity as suggested by Chesson et al. (1982) is highly possible. Importantly, the ratio of ADDL:ADL decreases with time of fermentation in the rumen. In contrast to KL, the ADL fraction is highly related to the extent of NDF digestion and true NDF indigestibility. The concentration of ADL within the NDF fraction determines the potentially digestible NDF pool size and accounts for nearly all of the variation in NDF indigestibility. The correlation of ADL with NDF digestion at early times of fermentation is poor and increases with time (Van Soest et al., 2005; Van Soest and Van Amburgh, 2006). Unlike the fraction measured as ADL, the soluble fraction (components of which are measured in ADDL, KL-ADL, or extractable phenolics) is more related to rate of NDF digestion. Van Soest et al. (2005) showed that rates and extents of digestion are accurately predicted using 24 h NDF factors and lignin content of NDF. The 24 h digestion probably reflects all the effects of the soluble phenolics, but that is one of the researchable problems. Chesson s model of cell wall digestion (Chesson, 1993) proposes that bacteria digest and release phenolic-carbohydrate fragments into solution within a microenvironment. Chesson s postulated phenolic-carbohydrate fragments would appear to be the fraction characterized by Lowry et al. (1994) as acid-detergent dispersible and also comprise some proportion of the L fraction described by Van Soest and Van Amburgh (2006). The phenolic acids and linked carbohydrate comprise the ADDL fraction described by Lowry et al. (1994). Use of an azo-dyeing technique allowed the authors to directly visualize the rate of color development during fermentation and relate it to immediately soluble lignin and some slower released lignin resulting from cleavage of ester bonds. It seems possible that the degree of crosslinking between phenolics and carbohydrate could influence the ease of microbial digestion and release of these fragments into solution. The ratio of ester:ether linkages and the steric hindrance resulting from these linkages could all influence the rate of cell wall fermentation. In fact, Lowry et al. (1994) proposed that ADDL is a spongy network of phenolic polymers left after acid detergent reflux and removal of hemicellulose. The ADDL fraction would represent the entire pool of lignified cell wall that is susceptible to cleavage in the rumen. Microbial dissolution of xylans would allow for some fraction of this pool to be dispersed into the ruminal fluid in soluble form. Finally, the research on lignin-carbohydrate chemistry would imply that the amount of ADDL actually degraded would be a function of the extent and type of linkages. The net result of this model is that the ADDL or L fraction would exert a controlling effect on the fast rate of NDF digestion and consequently on NDF digestion at early time points of fermentation. Due to chemical differences between grasses and legumes, the effect would be greater for grasses. For legumes, peptide linkages between carbohydrates and lignin may play a larger role (Van Soest and Van Amburgh, 2006). A

10 smaller ADDL fraction for legumes compared with grasses translates into a generally greater fast rate of NDF digestion with ADL exerting a greater negative effect on NDF digested. Crystallinity, enzyme accessible space, and other intrinsic plant factors presumably would also influence rate of NDF digestion for both grasses and legumes. PROPOSED STUDY TO TEST THE MODEL It appears reasonable to predict that a satisfactory model to explain the inhibitory effect of lignification on cell wall digestion would combine information from basic lignin chemistry studies with nutritional studies that have used detergent fractionation schemes. A proposed study has been outlined that will test key components of this model. The corn samples of varying NDF, ADL, and NDF digestibility described by Van Amburgh (2004) which include bmr as well as conventional hybrids will be combined with the bmr forage sorghum samples previously described. These samples would represent a wide range in lignin concentration, phenolic acid composition, and type of cross-linkages. Measurements of KL, ADL, L, absorbance at 280 nm to measure soluble lignin, and phenolic acid monomers and linkages would be conducted. The NDF fermentation kinetics would be measured and relationships between the measured lignin or lignin fractions would be investigated to determine the relative importance of the soluble lignin versus the core lignin on rate and extent of NDF digestion. REFERENCES Bout, S., and W. Vermerris A candidate-gene approach to clone the sorghum brown midrib gene encoding caffeic acid O-methyl-transferase. Mol. Genet. Gen. 269: Burritt, E. A., A. S. Bittner, J. C. Street, and M. J. Anderson Correlations of phenolic acids and xylose content of cell wall with in vitro dry matter digestibility of three maturing grasses. J. Dairy Sci. 67: Casler, M. D., and H. J. G. Jung Relationships of fibre, lignin, and phenolics to in vitro fibre digestibility in three perennial grasses. Anim. Feed Sci. Technol. 125: Cherney, D. J. R., J. H. Cherney, J. A. Patterson, and J. D. Axtell In vitro ruminal fiber digestion as influenced by phenolic-carbohydrate complexes released from sorghum cell walls. Anim. Feed Sci. Technol. 39: Chesson, A., C. S. Stewart, and R. J. Wallace Influence of plant phenolic acids on growth and cellulolytic activity of rumen bacteria. Appl. Environ. Microbiol. 44: Chesson, A Mechanistic models of forage cell wall degradation. Pages in Forage Cell Wall Structure and Digestibility. H. G. Jung, D. R. Buxton, R. D. Hatfield, and J. Ralph, ed. ASA-CSSA-SSSA, Madison, WI. Dann, H. M., K. W. Cotanch, R. J. Grant, S. R. Kramer, C. S. Ballard, and T. Takano Development of a chemical measure to better predict NDF digestibility for use in CPM-Dairy and other nutritional models. Report to ZEN-NOH National Federation of Agriculture Co-operative Associations, Tokyo, Japan.

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