Characterization and Elimination of Undesirable Protein Residues in Plant Cell Wall Materials for Enhancing Lignin Analysis by Solution-State Nuclear Magnetic Resonance Spectroscopy.

Protein polymers exist in every plant cell wall preparation, and they interfere with lignin characterization and quantification. Here, we report the structural characterization of the residual protein peaks in 2D NMR spectra in corn cob and kenaf samples and note that aromatic amino acids are ubiquitous and evident in spectra from various other plants and tissues. The aromatic correlations from amino acid residues were identified and assigned as phenylalanine and tyrosine. Phenylalanine's 3/5 correlation peak is superimposed on the peak from typical lignin p-hydroxyphenyl (H-unit) structures, causing an overestimation of the H units. Protein contamination also occurs when using cellulases to prepare enzyme lignins from virtually protein-free wood samples. We used a protease to remove the protein residues from the ball-milled cell walls, and we were able to reveal H-unit structures in lignins more clearly in the 2D NMR spectra, providing a better basis for their estimation.

Identification and suppression of the p-coumaroyl CoA:hydroxycinnamyl alcohol transferase in Zea mays L.

Grasses, such as Zea mays L. (maize), contain relatively high levels of p-coumarates (pCA) within their cell walls. Incorporation of pCA into cell walls is believed to be due to a hydroxycinnamyl transferase that couples pCA to monolignols. To understand the role of pCA in maize development, the p-coumaroyl CoA:hydroxycinnamyl alcohol transferase (pCAT) was isolated and purified from maize stems. Purified pCAT was subjected to partial trypsin digestion, and peptides were sequenced by tandem mass spectrometry. TBLASTN analysis of the acquired peptide sequences identified a single full-length maize cDNA clone encoding all the peptide sequences obtained from the purified enzyme. The cDNA clone was obtained and used to generate an RNAi construct for suppressing pCAT expression in maize. Here we describe the effects of suppression of pCAT in maize. Primary screening of transgenic maize seedling leaves using a new rapid analytical platform was used to identify plants with decreased amounts of pCA. Using this screening method, mature leaves from fully developed plants were analyzed, confirming reduced pCA levels throughout plant development. Complete analysis of isolated cell walls from mature transgenic stems and leaves revealed that lignin levels did not change, but pCA levels decreased and the lignin composition was altered. Transgenic plants with the lowest levels of pCA had decreased levels of syringyl units in the lignin. Thus, altering the levels of pCAT expression in maize leads to altered lignin composition, but does not appear to alter the total amount of lignin present in the cell walls.

Overexpression of a Sugarcane BAHD Acyltransferase Alters Hydroxycinnamate Content in Maize Cell Wall.

The purification of hydroxycinnamic acids [p-coumaric acid (pCA) and ferulic acid (FA)] from grass cell walls requires high-cost processes. Feedstocks with increased levels of one hydroxycinnamate in preference to the other are therefore highly desirable. We identified and conducted expression analysis for nine BAHD acyltransferase ScAts genes from sugarcane. The high conservation of AT10 proteins, together with their similar gene expression patterns, supported a similar role in distinct grasses. Overexpression of ScAT10 in maize resulted in up to 75% increase in total pCA content. Mild hydrolysis and derivatization followed by reductive cleavage (DFRC) analysis showed that pCA increase was restricted to the hemicellulosic portion of the cell wall. Furthermore, total FA content was reduced up to 88%, resulting in a 10-fold increase in the pCA/FA ratio. Thus, we functionally characterized a sugarcane gene involved in pCA content on hemicelluloses and generated a C4 plant that is promising for valorizing pCA production in biorefineries.

Grass lignin acylation: p-coumaroyl transferase activity and cell wall characteristics of C3 and C4 grasses.

Grasses are a predominant source of nutritional energy for livestock systems around the world. Grasses with high lignin content have lower energy conversion efficiencies for production of bioenergy either in the form of ethanol or to milk and meat through ruminants. Grass lignins are uniquely acylated with p-coumarates (pCA), resulting from the incorporation of monolignol p-coumarate conjugates into the growing lignin polymer within the cell wall matrix. The required acyl-transferase is a soluble enzyme (p-coumaroyl transferase, pCAT) that utilizes p-coumaroyl-CoenzymeA (pCA-CoA) as the activated donor molecule and sinapyl alcohol as the preferred acceptor molecule. Grasses (C3and C4) were evaluated for cell wall characteristics; pCA, lignin, pCAT activity, and neutral sugar composition. All C3 and C4 grasses had measurable pCAT activity, however the pCAT activities did not follow the same pattern as the pCA incorporation into lignin as expected.

Coniferyl ferulate incorporation into lignin enhances the alkaline delignification and enzymatic degradation of cell walls.

Incorporating ester interunit linkages into lignin could facilitate fiber delignification and utilization. In model studies with maize cell walls, we examined how partial substitution of coniferyl alcohol (a normal monolignol) with coniferyl ferulate (an ester conjugate from lignan biosynthesis) alters the formation and alkaline extractability of lignin and the enzymatic hydrolysis of structural polysaccharides. Coniferyl ferulate moderately reduced lignification and cell-wall ferulate copolymerization with monolignols. Incorporation of coniferyl ferulate increased lignin extractability by up to 2-fold in aqueous NaOH, providing an avenue for producing fiber with less noncellulosic and lignin contamination or of delignifying at lower temperatures. Cell walls lignified with coniferyl ferulate were more readily hydrolyzed with fibrolytic enzymes, both with and without alkaline pretreatment. Based on our results, bioengineering of plants to incorporate coniferyl ferulate into lignin should enhance lignocellulosic biomass saccharification and particularly pulping for paper production.

Three polyphenol oxidases from red clover (Trifolium pratense) differ in enzymatic activities and activation properties.

Polyphenol oxidases (PPOs) oxidize o-diphenols to o-quinones, which cause browning reactions in many wounded fruits, vegetables, and plants including the forage crop red clover (Trifolium pratense L.). Production of o-quinones in red clover inhibits postharvest proteolysis during the ensiling process. The cDNAs encoding three red clover PPOs were expressed individually in alfalfa (Medicago sativa L.), which lacks detectable endogenous foliar PPO activity and o-diphenols. Several physical and biochemical characteristics of the red clover PPOs in alfalfa extracts were determined. In transgenic alfalfa extracts, red clover PPOs exist in a latent state and are activated (10-40-fold increase in activity) by long incubations (>2 days) at ambient temperature or short incubations (<10 min) at > or =65 degrees C. PPO1 appears to be more stable at high temperatures than PPO2 or PPO3. During incubation at ambient temperature, the molecular masses of the PPO enzymes were reduced by approximately 20 kDa. The apparent pH optima of latent PPO1, PPO2, and PPO3 are 5.5, 6.9, and 5.1, respectively, and latent PPO1 is slightly activated (~5-fold) by low pH. Activation of the PPOs shifts the pH optima to approximately 7, and the activated PPOs retain substantial levels of activity as the pH increases above their optima. The latent and activated PPOs were surveyed for ability to oxidize various o-diphenols, and activation of the PPOs had little effect on substrate specificity. Activation increases the V max but not the affinity of the PPO enzymes for caffeic acid. Results indicate red clover PPOs undergo structural and kinetic changes during activation and provide new insights to their effects in postharvest physiology.

Cell wall composition in juvenile and adult leaves of maize (Zea mays L.).

Many leaf characteristics vary with position along the culm in maize (Zea mays L.) due to the existence of vegetative phase change and heteroblasty. The objective of this work was to determine if differences in cell wall composition exist among developmental phases and between Cg1, a developmental mutant, and wild-type maize. In one experiment, the middle third of fully elongated leaf blades from lower and upper regions of the shoot was harvested (midribs removed) and analyzed for several cell wall components. Averaged over five inbreds (De811, Ia5125, Mo17, P39, and Wh8584), lower leaf blades had higher levels of xylose and lower levels of total uronosyls, glucose, arabinose, and galactose (P < 0.05) than did upper leaf blades. With the exception of glucose, upper and lower leaves of Cg1 plants varied in the same manner as their near-isogenic siblings, except cell walls of Cg1 plants were more "juvenile" than cell walls of wild-type siblings at the same leaf stage. These data support the hypothesis that Cg1 delays but does not eliminate the transition from juvenile-vegetative to adult-vegetative phase. In a second experiment, juvenile (leaves 3 and 5), transition (leaf 7), and adult (leaves 9 and 11) leaves from inbreds B73 and De811 were harvested and analyzed as in the first experiment. As leaf number rose, total cell wall content of sample dry matter, total neutral sugars, glucose, xylose, and ester-linked monomers of p-coumaric acid and total ferulates including ferulate dimers increased linearly while total uronosyls acids, arabinose, and galactose declined linearly (P < 0.05). Glucose and xylose are major cell wall components released from cellulose and xylans after acid hydrolysis. Pectin, a minor component of grass cell walls, is composed of galacturonosyls, arabinose, and galactose. Secondary cell wall deposition increased between leaves 3 and 11 in a heteroblastic series, due to either increased cell wall content concomitant with decreased cell lumen size, changes in proportion of cell types (i.e., sclerenchyma), or a combination of these factors.

Grass Cell Walls: A Story of Cross-Linking.

Cell wall matrices are complex composites mainly of polysaccharides, phenolics (monomers and polymers), and protein. We are beginning to understand the synthesis of these major wall components individually, but still have a poor understanding of how cell walls are assembled into complex matrices. Valuable insight has been gained by examining intact components to understand the individual elements that make up plant cell walls. Grasses are a prominent group within the plant kingdom, not only for their important roles in global agriculture, but also for the complexity of their cell walls. Ferulate incorporation into grass cell wall matrices (C3 and C4 types) leads to a cross-linked matrix that plays a prominent role in the structure and utilization of grass biomass compared to dicot species. Incorporation of p-coumarates as part of the lignin structure also adds to the complexity of grass cell walls. Feruoylation results in a wall with individual hemicellulosic polysaccharides (arabinoxylans) covalently linked to each other and to lignin. Evidence strongly suggests that ferulates not only cross-link arabinoxylans, but may be important factors in lignification of the cell wall. Therefore, the distribution of ferulates on arabinoxylans could provide a means of structuring regions of the matrix with the incorporation of lignin and have a significant impact upon localized cell wall organization. The role of other phenolics in cell wall formation such as p-coumarates (which can have concentrations higher than ferulates) remains unknown. It is possible that p-coumarates assist in the formation of lignin, especially syringyl rich lignin. The uniqueness of the grass cell wall compared to dicot sepcies may not be so much in the gross composition of the wall, but how the distinctive individual components are organized into a functional wall matrix. These features are discussed and working models are provided to illustrate how changing the organization of feruoylation and p-coumaroylation could lead to differing cell wall properties.

Methyl esterification divergently affects the degradability of pectic uronosyls in nonlignified and lignified maize cell walls.

Nonlignified cell walls from Zea mays (L.) cell suspensions were incubated with and without pectin methylesterase (PME) and a portion were artificially lignified to assess how methyl esters influence the release of pectic uronosyls and total sugars from cell walls by fungal enzymes. Treatment with PME reduced uronosyl concentrations from 97 to 92 mg/g, reduced uronosyl methylation from 57% to 21%, and increased Klason lignin concentrations in artificially lignified cell walls from 99 to 116 mg/g. Although PME treatment slightly enhanced uronosyl release from nonlignified cell walls, it reduced uronosyl release from artificially lignified cell walls by 55% after 4 h and by 7% after 72 h of enzymatic hydrolysis. Pectin hydrolysis in PME treated cell walls was probably impaired by enhanced benzyl ester cross-linking of uronosyls to lignin via quinone methide intermediates. Variations in uronosyl methylation had little effect on the overall release of total sugars from cell walls.

Comparison of methods for glycogen analysis of in vitro fermentation pellets produced with strained ruminal inoculum.

Microbial glycogen measurement is used to account for fates of carbohydrate substrates. It is commonly applied to washed cells or pure cultures which can be accurately subsampled, allowing the use of smaller sample sizes. However, the nonhomogeneous fermentation pellets produced with strained rumen inoculum cannot be accurately subsampled, requiring analysis of the entire pellet. In this study, two microbial glycogen methods were compared for analysis of such fermentation pellets: boiling samples for 3h in 30% KOH (KOH) or for 15min in 0.2M NaOH (NaOH), followed by enzymatic hydrolysis with α-amylase and amyloglucosidase, and detection of released glucose. Total α-glucan was calculated as glucose×0.9. KOH and NaOH did not differ in the α-glucan detected in fermentation pellets (29.9 and 29.6mg, respectively; P=0.61). Recovery of different control α-glucans was also tested using KOH, NaOH, and a method employing 45min of bead beating (BB). For purified beef liver glycogen (water-soluble) recovery, BB (95.0%)>KOH (91.4%)>NaOH (87.4%; P<0.05), and for wheat starch (water-insoluble granules) recovery, NaOH (96.9%)>BB (93.8%)>KOH (91.0%; P<0.05). Recovery of isolated protozoal glycogen (water-insoluble granules) did not differ among KOH (87.0%), NaOH (87.6%), and BB (86.0%; P=0.81), but recoveries for all were below 90%. Differences among substrates in the need for gelatinization and susceptibility to destruction by alkali likely affected the results. In conclusion, KOH and NaOH glycogen methods provided comparable determinations of fermentation pellet α-glucan. The tests on purified α-glucans indicated that assessment of recovery in glycogen methods can differ by the control α-glucan selected.

Cell wall composition and digestibility alterations in Brachypodium distachyon achieved through reduced expression of the UDP-arabinopyranose mutase.

Nucleotide-activated sugars are essential substrates for plant cell-wall carbohydrate-polymer biosynthesis. The most prevalent grass cell wall (CW) sugars are glucose (Glc), xylose (Xyl), and arabinose (Ara). These sugars are biosynthetically related via the UDP-sugar interconversion pathway. We sought to target and generate UDP-sugar interconversion pathway transgenic Brachypodium distachyon lines resulting in CW carbohydrate composition changes with improved digestibility and normal plant stature. Both RNAi-mediated gene-suppression and constitutive gene-expression approaches were performed. CWs from 336 T0 transgenic plants with normal appearance were screened for complete carbohydrate composition. RNAi mutants of BdRGP1, a UDP-arabinopyranose mutase, resulted in large alterations in CW carbohydrate composition with significant decreases in CW Ara content but with minimal change in plant stature. Five independent RNAi-RGP1 T1 plant lines were used for in-depth analysis of plant CWs. Real-time PCR analysis indicated that gene expression levels for BdRGP1, BdRGP2, and BdRGP3 were reduced in RNAi-RGP1 plants to 15-20% of controls. CW Ara content was reduced by 23-51% of control levels. No alterations in CW Xyl and Glc content were observed. Corresponding decreases in CW ferulic acid (FA) and ferulic acid-dimers (FA-dimers) were observed. Additionally, CW p-coumarates (pCA) were decreased. We demonstrate the CW pCA decrease corresponds to Ara-coupled pCA. Xylanase-mediated digestibility of RNAi-RGP1 Brachypodium CWs resulted in a near twofold increase of released total carbohydrate. However, cellulolytic hydrolysis of CW material was inhibited in leaves of RNAi-RGP1 mutants. Our results indicate that targeted manipulation of UDP-sugar biosynthesis can result in biomass with substantially altered compositions and highlights the complex effect CW composition has on digestibility.

Structural and compositional modifications in lignin of transgenic alfalfa down-regulated in caffeic acid 3-O-methyltransferase and caffeoyl coenzyme A 3-O-methyltransferase.

Isolated lignins from alfalfa deficient in caffeic acid 3-O-methyltransferase contained benzodioxanes resulting from the incorporation of the novel monomer, 5-hydroxyconiferyl alcohol. Due to the high level incorporated into the soluble lignin fraction and the use of sensitive NMR instrumentation, unique structural features were revealed. A new type of end-unit, the 5-hydroxyguaiacyl glycerol unit, was identified. It was possible to establish that coniferyl alcohol, sinapyl alcohol, and the novel 5-hydroxyconiferyl alcohol can cross-couple with the 5-hydroxyguaiacyl units that are formed in the lignin, the latter giving rise to extended chains of benzodioxane units. There is also evidence that 5-hydroxyconiferyl alcohol couples with normal (guaiacyl or syringyl) lignin units. Lignin in the alfalfa deficient in caffeoyl CoA 3-O-methyltransferase was structurally similar to the control lignin but the transgenic exhibited a dramatic decrease in lignin content (approximately 20%) and modest increase in cellulose (approximately 10%) reflecting a 30% increase in cellulose:lignin ratio. The compositional changes in both transgenics potentially allow enhanced utilization of alfalfa as a major forage crop by increasing the digestibility of its stem fraction.

Comparison of the acetyl bromide spectrophotometric method with other analytical lignin methods for determining lignin concentration in forage samples.

Present analytical methods to quantify lignin in herbaceous plants are not totally satisfactory. A spectrophotometric method, acetyl bromide soluble lignin (ABSL), has been employed to determine lignin concentration in a range of plant materials. In this work, lignin extracted with acidic dioxane was used to develop standard curves and to calculate the derived linear regression equation (slope equals absorptivity value or extinction coefficient) for determining the lignin concentration of respective cell wall samples. This procedure yielded lignin values that were different from those obtained with Klason lignin, acid detergent acid insoluble lignin, or permanganate lignin procedures. Correlations with in vitro dry matter or cell wall digestibility of samples were highest with data from the spectrophotometric technique. The ABSL method employing as standard lignin extracted with acidic dioxane has the potential to be employed as an analytical method to determine lignin concentration in a range of forage materials. It may be useful in developing a quick and easy method to predict in vitro digestibility on the basis of the total lignin content of a sample.

Apoplastic pH and monolignol addition rate effects on lignin formation and cell wall degradability in maize.

Monolignol polymerization rate and apoplastic pH and may influence the formation of lignin and its interactions in cell walls. Primary maize walls were artificially lignified by gradual "end-wise" or rapid "bulk" polymerization of coniferyl alcohol at pH 4 or 5.5. Lignification efficiency was greatest for end-wise polymers at pH 5.5 (90-98%), intermediate for bulk polymers formed at either pH (54-82%), and lowest for end-wise polymers at pH 4 (41-53%). End-wise polymers had about 2.2-fold more ether inter-unit linkages and 70% fewer end-groups than bulk polymers. Low pH enhanced the formation of ether linkages in end-wise but not in bulk polymers. Differences in lignin structure did not influence the enzymatic degradability of cell walls, but lowering apoplastic pH from 5.5 to 4.0 during lignification reduced cell wall degradability by 25%. Further studies indicated this pH-dependent depression in degradability was related to cell wall cross-links formed via lignin quinone methide intermediates.

Model studies of ferulate-coniferyl alcohol cross-product formation in primary maize walls: implications for lignification in grasses.

Ferulate and diferulates mediate cell wall cross-linking in grasses, but little is known about their cross-coupling reactions with monolignols and their role in lignin formation in primary cell walls. Feruloylated primary walls of maize were artificially lignified and then saponified to release ferulate and diferulates and their cross-products with coniferyl alcohol for analysis by GC-FID, GC-MS, and NMR spectroscopy. Ferulate and 5-5-coupled diferulate had a greater propensity than 8-coupled diferulates to copolymerize with coniferyl alcohol, forming mostly 4-O-beta' and 8-beta' and some 8-O-4' and 8-5' cross-coupled structures. Some 8-beta' structures de-esterified from xylans, but these cross-links were subsequently replaced as 8-coupled diferulates formed stable cross-coupled structures with lignin. Based on the incorporation kinetics of ferulate and diferulates and the predicted growth of lignin, cross-products formed at the onset of lignification acted as nucleation sites for lignin polymerization.

Lignins and ferulate-coniferyl alcohol cross-coupling products in cereal grains.

Plant cell walls containing suberin or lignin in the human diet are conjectured to protect against colon cancer. To confirm the existence of authentic lignin in cereal grain dietary fibers, the DFRC (derivatization followed by reductive cleavage) method was applied to different cereal grain dietary fibers. By cleavage of diagnostic arylglycerol-beta-aryl (beta-O-4) ether linkages and identification of the liberated monolignols, it was ascertained that lignins are truly present in cereal grains. From the ratios of the liberated monolignols coniferyl alcohol and sinapyl alcohol, it is suggested that lignin compositions vary among cereals. Furthermore, dimeric cross-coupling products, comprising ferulate and coniferyl alcohol, were identified in most cereal fibers investigated. These ferulate 4-O-beta- and 8-beta-coniferyl alcohol cross-coupled structures indicate radical cross-coupling of polysaccharides to lignin precursors via ferulate.

Variations in the cell wall composition of maize brown midrib mutants.

Most studies published thus far on the four brown midrib (bm) mutants (bm1, bm2, bm3, and bm4) in maize (Zea mays L.) have focused on one or two individual mutants, and comparisons between studies have been difficult because of variation in genetic backgrounds, maturity, and source of tissue. Detailed analyses of the stalks of the four bm single mutants and a bm1-bm2 double mutant in a common genetic background (inbred A619) revealed structural and compositional changes in their isolated cell walls and lignins compared to the wild-type inbred. 2D-NMR revealed a significant presence of benzodioxane units in the bm3 isolated lignin. 1D (13)C NMR revealed increased aldehyde levels in the bm1 and bm1-bm2 mutants compared to the wild-type inbred. The bm3 and bm1-bm2 mutants contained less Klason lignin in the isolated cell walls. The bm1, bm3, and bm1-bm2 mutants contained approximately 50% less esterified p-coumaric acid with noticeably elevated levels of ferulate in the bm3 mutant. A difference among bm mutants in the solubility of p-coumaric acid-lignin complexes during cellulase enzyme treatment was also discovered, suggesting that the bm mutations might also differ in the structural organization of lignin.

Chemical composition and enzymatic degradability of xylem and nonxylem walls isolated from alfalfa internodes.

During plant maturation, degradability of alfalfa (Medicago sativa L.) stems declines due to accumulation of highly lignified xylary tissue. Xylem and nonxylem tissues dissected from lower alfalfa internodes were analyzed for cell wall constituents and degradability. Cell walls comprised 740 mg g(-1) of xylem and 533 mg g(-1) of nonxylem tissues. Xylem tissues contributed about 60% of the cell wall mass in internodes. Xylem walls contained 28% lignin, 4% pectin, 29% hemicellulose, and 39% cellulose as compared to 15% lignin, 25% pectin, 30% hemicellulose, and 30% cellulose in nonxylem walls. Fungal enzymes hydrolyzed 22 and 73% of the structural carbohydrates in xylem and nonxylem walls, respectively. In both cell wall fractions, the release of xylose was 56-90% lower than that of other sugars, indicating that lignin preferentially restricted xylan degradation in secondary walls and xyloglucan degradation in primary walls. Elucidation of lignin-xylose interactions may reveal strategies for improving fiber degradability of alfalfa.

Sinapate dehydrodimers and sinapate-ferulate heterodimers in cereal dietary fiber.

Two 8-8-coupled sinapic acid dehydrodimers and at least three sinapate-ferulate heterodimers have been identified as saponification products from different insoluble and soluble cereal grain dietary fibers. The two 8-8-disinapates were authenticated by comparison of their GC retention times and mass spectra with authentic dehydrodimers synthesized from methyl or ethyl sinapate using two different single-electron metal oxidant systems. The highest amounts (481 microg/g) were found in wild rice insoluble dietary fiber. Model reactions showed that it is unlikely that the dehydrodisinapates detected are artifacts formed from free sinapic acid during the saponification procedure. The dehydrodisinapates presumably derive from radical coupling of sinapate-polymer esters in the cell wall; the radical coupling origin is further confirmed by finding 8-8 and 8-5 (and possibly 8-O-4) sinapate-ferulate cross-products. Sinapates therefore appear to have an analogous role to ferulates in cross-linking polysaccharides in cereal grains and presumably grass cell walls in general.

Improving ethanol production from alfalfa stems via ambient-temperature acid pretreatment and washing.

The concept of co-production of liquid fuel (ethanol) along with animal feed on farm was proposed, and the strategy of using ambient-temperature acid pretreatment, ensiling and washing to improve ethanol production from alfalfa stems was investigated. Alfalfa stems were separated and pretreated with sulfuric acid at ambient-temperature after harvest, and following ensiling, after which the ensiled stems were subjected to simultaneous saccharification and fermentation (SSF) for ethanol production. Ethanol yield was improved by ambient-temperature sulfuric acid pretreatment before ensiling, and by washing before SSF. It was theorized that the acid pretreatment at ambient temperature partially degraded hemicellulose, and altered cell wall structure, resulted in improved cellulose accessibility, whereas washing removed soluble ash in substrates which could inhibit the SSF. The pH of stored alfalfa stems can be used to predict the ethanol yield, with a correlation coefficient of +0.83 for washed alfalfa stems.