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.

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.

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.

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.

Sequencing around 5-hydroxyconiferyl alcohol-derived units in caffeic acid O-methyltransferase-deficient poplar lignins.

Caffeic acid O-methyltransferase (COMT) is a bifunctional enzyme that methylates the 5- and 3-hydroxyl positions on the aromatic ring of monolignol precursors, with a preference for 5-hydroxyconiferaldehyde, on the way to producing sinapyl alcohol. Lignins in COMT-deficient plants contain benzodioxane substructures due to the incorporation of 5-hydroxyconiferyl alcohol (5-OH-CA), as a monomer, into the lignin polymer. The derivatization followed by reductive cleavage method can be used to detect and determine benzodioxane structures because of their total survival under this degradation method. Moreover, partial sequencing information for 5-OH-CA incorporation into lignin can be derived from detection or isolation and structural analysis of the resulting benzodioxane products. Results from a modified derivatization followed by reductive cleavage analysis of COMT-deficient lignins provide evidence that 5-OH-CA cross couples (at its beta-position) with syringyl and guaiacyl units (at their O-4-positions) in the growing lignin polymer and then either coniferyl or sinapyl alcohol, or another 5-hydroxyconiferyl monomer, adds to the resulting 5-hydroxyguaiacyl terminus, producing the benzodioxane. This new terminus may also become etherified by coupling with further monolignols, incorporating the 5-OH-CA integrally into the lignin structure.

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.

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.

Effects of coumarate 3-hydroxylase down-regulation on lignin structure.

Down-regulation of the gene encoding 4-coumarate 3-hydroxylase (C3H) in alfalfa massively but predictably increased the proportion of p-hydroxyphenyl (P) units relative to the normally dominant guaiacyl (G) and syringyl (S) units. Stem levels of up to approximately 65% P (from wild-type levels of approximately 1%) resulting from down-regulation of C3H were measured by traditional degradative analyses as well as two-dimensional 13C-1H correlative NMR methods. Such levels put these transgenics well beyond the P:G:S compositional bounds of normal plants; p-hydroxyphenyl levels are reported to reach a maximum of 30% in gymnosperm severe compression wood zones but are limited to a few percent in dicots. NMR also revealed structural differences in the interunit linkage distribution that characterizes a lignin polymer. Lower levels of key beta-aryl ether units were relatively augmented by higher levels of phenylcoumarans and resinols. The C3H-deficient alfalfa lignins were devoid of beta-1 coupling products, highlighting the significant differences in the reaction course for p-coumaryl alcohol versus the two normally dominant monolignols, coniferyl and sinapyl alcohols. A larger range of dibenzodioxocin structures was evident in conjunction with an approximate doubling of their proportion. The nature of each of the structural units was revealed by long range 13C-1H correlation experiments. For example, although beta-ethers resulted from the coupling of all three monolignols with the growing polymer, phenylcoumarans were formed almost solely from coupling reactions involving p-coumaryl alcohol; they resulted from both coniferyl and sinapyl alcohol in the wild-type plants. Such structural differences form a basis for explaining differences in digestibility and pulping performance of C3H-deficient plants.

Genetic distance and its association with heterosis in cacao

The efficiency of cacao breeding program can be increased by choosing superior crosses to be made between divergent clones. We assessed the genetic distance among five clones with RAPD data (genetic distance - GD) and with yield component data (Mahalanobis distance - MD). The clones were evaluated in a diallel, during five years, for five yield components. A total of 130 RAPD bands were scored. GD and MD were used to determine the correlation between genetic distances among clones and the performance of their hybrids. The correlation between GD and MD was 0.67 (P=0.03). Both distances were related to heterotic performance of hybrids for wet seed weight/plant and wet seed weight/fruit. The average hybrid performance for the same two yield components was correlated with only MD. Hence, genetic distances measured by RAPD and yield components can be used as a guide to the choice of the superior crosses

Combinatorial modification of multiple lignin traits in trees through multigene cotransformation.

Lignin quantity and reactivity [which is associated with its syringyl/guaiacyl (S/G) constituent ratio] are two major barriers to wood-pulp production. To verify our contention that these traits are regulated by distinct monolignol biosynthesis genes, encoding 4-coumarate-CoA ligase (4CL) and coniferaldehyde 5-hydroxylase (CAld5H), we used Agrobacterium to cotransfer antisense 4CL and sense CAld5H genes into aspen (Populus tremuloides). Trees expressing each one and both of the transgenes were produced with high efficiency. Lignin reduction by as much as 40% with 14% cellulose augmentation was achieved in antisense 4CL plants; S/G-ratio increases as much as 3-fold were observed without lignin quantity change in sense CAld5H plants. Consistent with our contention, these effects were independent but additive, with plants expressing both transgenes having up to 52% less lignin, a 64% higher S/G ratio, and 30% more cellulose. An S/G-ratio increase also accelerated cell maturation in stem secondary xylem, pointing to a role for syringyl lignin moieties in coordinating xylem secondary wall biosynthesis. The results suggest that this multigene cotransfer system should be broadly useful for plant genetic engineering and functional genomics.

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.

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.

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.