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.

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.

Extraction, composition, and functional properties of dried alfalfa (Medicago sativa L.) leaf protein.

BACKGROUND: Alfalfa is considered a potential feedstock for biofuels; co-products with value-added uses would enhance process viability. This work evaluated dried alfalfa leaves for protein production and describes the functional properties of the protein. RESULTS: Dried alfalfa leaves contained 260 g kg-1 dry basis (DB) crude protein, with albumins being the major fraction (260 g kg-1 of total protein). Alkali solubilization for 2 h at 50 °C, acid precipitation, dialysis, and freeze-drying produced a protein concentrate (600 g kg-1 DB crude protein). Alfalfa leaf protein concentrate showed moderate solubility (maximum 500 g kg-1 soluble protein from pH 5.5 to 10), excellent emulsifying properties (activity 158-219 m2 g-1 protein, stability 17-49 min) and minimal loss of solubility during heating at pH ≥ 7.0. CONCLUSIONS: It is technically feasible to extract protein with desirable emulsifying and heat stability properties from dried alfalfa leaves; however, the dried form may not be a practical starting material for protein production, given the difficulty of achieving high yields and high-purity protein product. © 2016 Society of Chemical Industry.

Cell wall chemical characteristics of whole-crop cereal silages harvested at three maturity stages.

BACKGROUND: In cooler climates such as found in Scandinavian countries cereals are important feedstuffs for ruminants often ensiled as whole-crop cereal silages (WCCS) to preserve nutrients. Animal performance varies with the type of cereal forage and stage of cereal development being ensiled. Cell wall isolation and analysis was undertaken to determine differences among cereal silages harvested at different stages of maturity. RESULTS: A set of 27 WCCS samples of barley, wheat and oats harvested at heading, early milk, and dough stages of maturity were analyzed for cell wall (CW) composition and compared to previous NDF analyses. Total CW concentrations of the WCCS were higher than the NDF concentration. The lignin concentration was higher (P < 0.001) in oats (111 g kg(-1) DM) than in barley (88 g kg(-1) DM) and wheat (91 g kg(-1) DM). Ferulates (ester and ether linked) ranged from 12.2 to 14.9 g kg(-1) across forage types and maturity stages. The correlation between total cell wall xylose and HC concentrations (NDF-ADF) was lower than expected in all forages (R = 0.63). CONCLUSION: The more comprehensive analyses of cell walls provide detailed composition of the different WCCS that vary due to the maturity and type of cereal. © 2016 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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.

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.

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.

Novel entries in a fungal biofilm matrix encyclopedia.

Virulence of Candida is linked with its ability to form biofilms. Once established, biofilm infections are nearly impossible to eradicate. Biofilm cells live immersed in a self-produced matrix, a blend of extracellular biopolymers, many of which are uncharacterized. In this study, we provide a comprehensive analysis of the matrix manufactured by Candida albicans both in vitro and in a clinical niche animal model. We further explore the function of matrix components, including the impact on drug resistance. We uncovered components from each of the macromolecular classes (55% protein, 25% carbohydrate, 15% lipid, and 5% nucleic acid) in the C. albicans biofilm matrix. Three individual polysaccharides were identified and were suggested to interact physically. Surprisingly, a previously identified polysaccharide of functional importance, ß-1,3-glucan, comprised only a small portion of the total matrix carbohydrate. Newly described, more abundant polysaccharides included α-1,2 branched α-1,6-mannans (87%) associated with unbranched ß-1,6-glucans (13%) in an apparent mannan-glucan complex (MGCx). Functional matrix proteomic analysis revealed 458 distinct activities. The matrix lipids consisted of neutral glycerolipids (89.1%), polar glycerolipids (10.4%), and sphingolipids (0.5%). Examination of matrix nucleic acid identified DNA, primarily noncoding sequences. Several of the in vitro matrix components, including proteins and each of the polysaccharides, were also present in the matrix of a clinically relevant in vivo biofilm. Nuclear magnetic resonance (NMR) analysis demonstrated interaction of aggregate matrix with the antifungal fluconazole, consistent with a role in drug impedance and contribution of multiple matrix components. Importance: This report is the first to decipher the complex and unique macromolecular composition of the Candida biofilm matrix, demonstrate the clinical relevance of matrix components, and show that multiple matrix components are needed for protection from antifungal drugs. The availability of these biochemical analyses provides a unique resource for further functional investigation of the biofilm matrix, a defining trait of this lifestyle.

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.

Cell wall composition throughout development for the model grass Brachypodium distachyon.

Temperate perennial grasses are important worldwide as a livestock nutritive energy source and a potential feedstock for lignocellulosic biofuel production. The annual temperate grass Brachypodium distachyon has been championed as a useful model system to facilitate biological research in agriculturally important temperate forage grasses based on phylogenetic relationships. To physically corroborate genetic predictions, we determined the chemical composition profiles of organ-specific cell walls throughout the development of two common diploid accessions of Brachypodium distachyon, Bd21-3 and Bd21. Chemical analysis was performed on cell walls isolated from distinct organs (i.e., leaves, sheaths, stems, and roots) at three developmental stages of (1) 12-day seedling, (2) vegetative-to-reproductive transition, and (3) mature seed fill. In addition, we have included cell wall analysis of embryonic callus used for genetic transformations. Composition of cell walls based on components lignin, hydroxycinnamates, uronosyls, neutral sugars, and protein suggests that Brachypodium distachyon is similar chemically to agriculturally important forage grasses. There were modest compositional differences in hydroxycinnamate profiles between accessions Bd21-3 and Bd21. In addition, when compared to agronomical important C3 grasses, more mature Brachypodium stem cell walls have a relative increase in glucose of 48% and a decrease in lignin of 36%. Though differences exist between Brachypodium and agronomical important C3 grasses, Brachypodium distachyon should be still a useful model system for genetic manipulation of cell wall composition to determine the impact upon functional characteristics such as rumen digestibility or energy conversion efficiency for bioenergy production.

Proteomic analysis of cell walls of two developmental stages of alfalfa stems.

Cell walls are important for the growth and development of all plants. They are also valuable resources for feed and fiber, and more recently as a potential feedstock for bioenergy production. Cell wall proteins comprise only a fraction of the cell wall, but play important roles in establishing the walls and in the chemical interactions (e.g., crosslinking) of cell wall components. This crosslinking provides structure, but restricts digestibility of cell wall complex carbohydrates, limiting available energy in animal and bioenergy production systems. Manipulation of cell wall proteins could be a strategy to improve digestibility. An analysis of the cell wall proteome of apical alfalfa stems (less mature, more digestible) and basal alfalfa stems (more mature, less digestible) was conducted using a recently developed low-salt/density gradient method for the isolation of cell walls. Walls were subsequently subjected to a modified extraction utilizing EGTA to remove pectins, followed by a LiCl extraction to isolate more tightly bound proteins. Recovered proteins were identified using shotgun proteomics. We identified 272 proteins in the alfalfa stem cell wall proteome, 153 of which had not previously been identified in cell wall proteomic analyses. Nearly 70% of the identified proteins were predicted to be secreted, as would be expected for most cell wall proteins, an improvement over previously published studies using traditional cell wall isolation methods. A comparison of our and several other cell wall proteomic studies indicates little overlap in identified proteins among them, which may be largely due to differences in the tissues used as well as differences in experimental approach.

Sodium hydroxide pretreatment of genetically modified switchgrass for improved enzymatic release of sugars.

Overcoming biomass recalcitrance to bioconversion is crucial for cellulosic biofuels commercialization. In this study, Alamo switchgrass (Panicum virgatum L.) was genetically transformed to suppress the expression of 4-coumarate-CoA ligase (4CL). The transgenic plants were determined to have lignin content reductions of up to 5.8%. The ratios of acid soluble lignin (ASL) to acid insoluble lignin (AIL) and syringyl/guaiacyl (S/G) in transgenic plants were 21.4-64.3% and 11.8-164.5%, respectively, higher than those of conventional biomass. Both conventional and transgenic plants were pretreated with 0.5%, 1%, and 2% (w/v) NaOH for 15, 30, and 60min at 121°C, followed by enzymatic hydrolysis with commercial cellulases and xylanases. At the optimal conditions, the glucan and xylan conversion efficiency in the best transgenic plants were 16% and 18% higher than the conventional plant, respectively. The results show that down-regulation of 4CL gene promoted enzymatic hydrolysis of plant cell walls following a mild alkali pretreatment.

Optimizing on-farm pretreatment of perennial grasses for fuel ethanol production.

Switchgrass (Panicum virgatum L.) and reed canarygrass (Phalaris arundinacea L.) were pretreated under ambient temperature and pressure with sulfuric acid and calcium hydroxide in separate experiments. Chemical loadings from 0 to 100g (kg DM)(-1) and durations of anaerobic storage from 0 to 180days were investigated by way of a central composite design at two moisture contents (40% or 60% w.b.). Pretreated and untreated samples were fermented to ethanol by Saccharomyces cerevisiae D5A in the presence of a commercially available cellulase (Celluclast 1.5L) and beta-glucosidase (Novozyme 188). Xylose levels were also measured following fermentation because xylose is not metabolized by S. cerevisiae. After sulfuric acid pretreatment and anaerobic storage, conversion of cell wall glucose to ethanol for reed canarygrass ranged from 22% to 83% whereas switchgrass conversions ranged from 16% to 46%. Pretreatment duration had a positive effect on conversion but was mitigated with increased chemical loadings. Conversions after calcium hydroxide pretreatment and anaerobic storage ranged from 21% to 55% and 18% to 54% for reed canarygrass and switchgrass, respectively. The efficacy of lime pretreatment was found to be highly dependent on moisture content. Moreover, pretreatment duration was only found to be significant for reed canarygrass. Although significant levels of acetate and lactate were observed in the biomass after storage, S. cerevisiae was not found to be inhibited at a 10% solids loading.

In vitro proteolytic inhibition, polyphenol oxidase activity, and soluble o-diphenols in grasses and cereals.

Producing high quality forage remains a challenge due to potential protein degradation during ensiling. Ten grass species and four cereals were screened for proteolytic inhibition, polyphenol oxidase (PPO) activity, and the presence of soluble PPO substrates. In orchardgrass, ryegrass, smooth bromegrass, and meadow fescue extracts, the addition of caffeic acid resulted in 70-98% reduction in proteolysis for both greenhouse and field grown environments. These same grass extracts exhibited 10- to 1000-fold higher PPO activity compared to all other grasses and cereals, depending on the substrate supplied and environment grown. Chlorogenic acid and caffeic acid were the two abundant soluble o-diphenols. Tall fescue and timothy exhibiting low PPO activity contained the highest levels of chlorogenic acid (3-11 micromol g(-1) FW): 10- to 100-fold more than any o-diphenol in all other grasses. These results suggest that several grass species contain PPO activity, but may lack appropriate o-diphenol substrates to effectively inhibit proteolysis during ensiling while other grasses lack PPO activity but contain potential soluble substrate. Environmental factors appear to influence these parameters, creating an opportunity to exploit and enhance proteolytic inhibition during ensiling by genetic manipulation.

Comparing corn types for differences in cell wall characteristics and p-coumaroylation of lignin.

This study was undertaken to compare cell wall characteristics including levels of p-coumarate (pCA) and lignin in corn (Zea mays L.) types. Five different types of corn, four commercial and Teosinte, were grown in the greenhouse in individual pots. For each corn type replicate stems were harvested at tassel emergence. Tissues for cell wall analysis were harvested from stems (separated into rind and pith tissues) and roots. Stem cell wall characteristics across the different corn types were similar for total neutral sugars, total uronosyls, lignin, and phenolic acids. However, the neutral sugar composition of root cell walls was markedly different, with high levels of galactose and arabinose. Levels of pCA in the different tissues ranged from 13.8 to 33.1 mg g(-1) of CW depending upon the type of tissue. There was no evidence that pCA was incorporated into cell walls attached to arabinoxylans. Lignin levels were similar within a given tissue, with pith ranging from 86.1 to 132.0 mg g(-1) of CW, rind from 178.4 to 236.6 mg g(-1) of CW, and roots from 216.5 to 242.6 mg g(-1) of CW. The higher values for lignins in root tissue may be due to suberin remaining in the acid-insoluble residue, forming Klason lignins. With the exception of root tissues, higher pCA levels accompanied higher lignin levels. This may indicate a potential role of pCA aiding lignin formation in corn cell walls during the lignification process.

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.

A potential role for sinapyl p-coumarate as a radical transfer mechanism in grass lignin formation.

Grass lignins are differentiated from other lignin types by containing relatively large amounts of p-coumaric acid (pCA) acylating the C-9 position of lignin subunits. In the case of a mature corn (Zea mays L.) stems, pCA constitutes 15-18% of a dioxane soluble enzyme lignin. The major portion of the pCA is specifically attached to syringyl residues. Studies with isolated corn wall peroxidases show that pCA readily undergoes radical coupling in the presence of hydrogen peroxide, whereas sinapyl alcohol radical coupling proceeds more slowly. Analysis of corn wall peroxidases did not reveal specific enzymes that would lead to the preferred incorporation of sinapyl alcohol as seen in other plants. The addition of ethyl ferulate, methyl p-coumarate, or sinapyl p-coumarate conjugates to a reaction mixture containing peroxidase, sinapyl alcohol, and hydrogen peroxide stimulated the rate of sinapyl alcohol radical coupling by 10-20-fold. Based on spectral analysis it appears that pCA and ferulate radicals form rapidly, but the radical is readily transferred to sinapyl alcohol. The newly formed sinapyl alcohol radicals undergo coupling and cross-coupling reactions. However, sinapyl alcohol radicals do not cross-couple with pCA radicals. As long as hydrogen peroxide is limiting pCA remains uncoupled. Ferulates have similar reaction patterns in terms of radical transfer though they appear to cross-couple in the reaction mixture more readily then pCA. The role of pCA may be to internally provide a radical transfer mechanism for optimizing radical coupling of sinapyl alcohol into the growing lignin polymer. Attachment of some pCA to sinapyl alcohol ensures localization of the radical transfer mechanism in areas where sinapyl alcohol is being incorporated into lignin.

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.