Structural characterization of alkaline hydrogen peroxide pretreated grasses exhibiting diverse lignin phenotypes

Background

For cellulosic biofuels processes, suitable characterization of the lignin remaining within the cell wall and correlation of quantified properties of lignin to cell wall polysaccharide enzymatic deconstruction is underrepresented in the literature. This is particularly true for grasses which represent a number of promising bioenergy feedstocks where quantification of grass lignins is particularly problematic due to the high fraction of p-hydroxycinnamates. The main focus of this work is to use grasses with a diverse range of lignin properties, and applying multiple lignin characterization platforms, attempt to correlate the differences in these lignin properties to the susceptibility to alkaline hydrogen peroxide (AHP) pretreatment and subsequent enzymatic deconstruction.

Results

We were able to determine that the enzymatic hydrolysis of cellulose to to glucose (i.e. digestibility) of four grasses with relatively diverse lignin phenotypes could be correlated to total lignin content and the content of p-hydroxycinnamates, while S/G ratios did not appear to contribute to the enzymatic digestibility or delignification. The lignins of the brown midrib corn stovers tested were significantly more condensed than a typical commercial corn stover and a significant finding was that pretreatment with alkaline hydrogen peroxide increases the fraction of lignins involved in condensed linkages from 88?C95% to ~99% for all the corn stovers tested, which is much more than has been reported in the literature for other pretreatments. This indicates significant scission of ??-O-4 bonds by pretreatment and/or induction of lignin condensation reactions. The S/G ratios in grasses determined by analytical pyrolysis are significantly lower than values obtained using either thioacidolysis or 2DHSQC NMR due to presumed interference by ferulates.

Conclusions

It was found that grass cell wall polysaccharide hydrolysis by cellulolytic enzymes for grasses exhibiting a diversity of lignin structures and compositions could be linked to quantifiable changes in the composition of the cell wall and properties of the lignin including apparent content of the p-hydroxycinnamates while the limitations of S/G estimation in grasses is highlighted.     

Relationships of fibre,lignin, and phenolics to in vitro fibre digestibility in three perennial grasses

Livestock performance can be improved by increasing the digestibility of feeds, one of the key elements of nutritional quality. Digestibility of feeds can be improved by breeding forage crops with modified cell-wall structure, increasing the potential availability of energy from the cell wall to rumen microbes and livestock. The objectives of this research were to identify interrelationships among lignin and phenolic components of the fibre fraction of three perennial grasses and to determine their influence on in vitro fibre digestibility. Differences in etherified and esterified ferulate and esterified p-coumarate among clones of three perennial grasses were generally repeatable across harvests. The concentration of neutral detergent fibre (NDF) and Klason lignin within the NDF fraction were the factors most limiting to 24-h in vitro digestibility, with NDF being the most important. Klason lignin and etherified ferulate were the factors most limiting to 96-h in vitro digestibility for all three species. Due to its positive correlation with NDF, selection for low etherified ferulate should be avoided in smooth bromegrass (Bromus inermis Leyss) and cockfsoot (Dactylis glomerata L.). It should be possible to select and breed for low concentrations of lignin to improve digestibility without decreasing NDF in these two species. However, in reed canarygrass (Phalaris arundinacea L.), both lignin and etherified ferulate were positively correlated with NDF, indicating that selection for increased digestibility should be based directly on some measure of in vitro digestibility to avoid the fitness problems associated with reduced NDF.     

Improvement of biomass through lignin modification

Lignin, a major component of the cell wall of vascular plants, has long been recognized for its negative impact on forage quality, paper manufacturing, and, more recently, cellulosic biofuel production. Over the last two decades, genetic and biochemical analyses of brown midrib mutants of maize, sorghum and related grasses have advanced our understanding of the relationship between lignification and forage digestibility. This work has also inspired genetic engineering efforts aimed at generating crops with altered lignin, with the expectation that these strategies would enhance forage digestibility and/or pulping efficiency. The knowledge gained from these bioengineering efforts has greatly improved our understanding of the optimal lignin characteristics required for various applications of lignocellulosic materials while also contributing to our understanding of the lignin biosynthetic pathway. The recent upswing of interest in cellulosic biofuel production has become the new focus of lignin engineering. Populus trichocarpa and Brachypodium distachyon are emerging as model systems for energy crops. Lignin research on these systems, as well as on a variety of proposed energy crop species, is expected to shed new light on lignin biosynthesis and its regulation in energy crops, and lead to rational genetic engineering approaches to modify lignin for improved biofuel production.     

Improvement of in-rumen digestibility of alfalfa forage by genetic manipulation of lignin O-methyltransferases

Lignin inhibits forage digestibility by ruminant animals, and lignin levels and the proportion of dimethylated syringyl (S) lignin monomers increase with progressive maturity in stems of forage crops. We generated transgenic alfalfa (Medicago sativa L.) with reduced lignin content and altered lignin composition. Down-regulation of caffeic acid 3-O-methyltransferase (COMT) reduces lignin content, accompanied by near total loss of S lignin, whereas down-regulation of caffeoyl coenzyme A 3-O-methyltransferase (CCoAOMT) reduces lignin content without reduction in S lignin. These changes are not accompanied by altered ratios of cell wall polysaccharides. Analysis of rumen digestibility of alfalfa forage in fistulated steers revealed improved digestibility of forage from COMT down-regulated plants, but a greater improvement in digestibility following down-regulation of CCoAOMT. The results indicate that both lignin content and composition affect digestibility of alfalfa forage, and reveal a new strategy for forage quality improvement by genetic manipulation of CCoAOMT expression.     

Characterization of novel Brown midrib 6 mutations affecting lignin biosynthesis in sorghum

The presence of lignin reduces the quality of lignocellulosic biomass for forage materials and feedstock for biofuels. In C4 grasses,the brown midrib phenotype has been linked to mutations to genes in the monolignol biosynthesis pathway. For example,the Bmr6 gene in sorghum(Sorghum bicolor) has been previously shown to encode cinnamyl alcohol dehydrogenase(CAD),which catalyzes the final step of the monolignol biosynthesis pathway. Mutations in this gene have been shown to reduce the abundance of lignin,enhance digestibility,and improve saccharification efficiencies and ethanol yields. Nine sorghum lines harboring five different bmr6 alleles were identified in an EMS-mutagenized TILLING population. DNA sequencing of Bmr6 revealed that the majority of the mutations impacted evolutionarily conserved amino acids while three-dimensional structural modeling predicted that all of these alleles interfered with the enzyme's ability to bind with its NADPH cofactor. All of the new alleles reduced in vitro CAD activity levels and enhanced glucose yields following saccharification. Further,many of these lines were associated with higher reductions in acid detergent lignin compared to lines harboring the previously characterized bmr6-ref allele. These bmr6 lines represent new breeding tools for manipulating biomass composition to enhance forage and feedstock quality.     

Improvement of cell wall digestibility in tall fescue by <Emphasis Type="Italic">Oryza sativa</Emphasis> SECONDARY WALL NAC DOMAIN PROTEIN2 chimeric repressor

Forage digestibility is one of the most important factors in livestock performance. As grasses grow and mature, dry matter increases but they become fibrous with secondary cell wall deposition and lignification of sclerenchyma cells, and forage quality drops. In rice (Oryza sativa), the SECONDARY WALL NAC DOMAIN PROTEIN2 fused with the modified EAR-like motif repression domain (OsSWN2-SRDX) reduces secondary cell wall thickening in sclerenchyma cells. We introduced OsSWN2-SRDX under the control of the OsSWN1 promoter into tall fescue (Festuca arundinacea Schreb.) to increase cell wall digestibility. Of 23 transgenic plants expressing OsSWN2-SRDX, nine had brittle internodes that were easily broken by bending. Their secondary cell walls were significantly thinner than those of the wild type in interfascicular fibers of internodes and in cortical fiber cells between leaf epidermal cells and vascular bundles. The dry matter digestibility increased by 11.8% in stems and by 6.8% in leaves compared with the wild type, and therefore forage quality was improved. In stem interfascicular fibers, acid detergent fiber and acid insoluble lignin were greatly reduced. Thus, the reduction of indigestible fiber composed of cellulose and lignin increased the degradability of sclerenchyma cell walls. OsSWN2-SRDX plants offer great potential in the genetic improvement of forage digestibility.     

Functional analyses of caffeic acid O-Methyltransferase and Cinnamoyl-CoA-reductase genes from perennial ryegrass (Lolium perenne)

Cinnamoyl CoA-reductase (CCR) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the biosynthesis of monolignols, which serve as building blocks in the formation of plant lignin. We identified candidate genes encoding these two enzymes in perennial ryegrass (Lolium perenne) and show that the spatio-temporal expression patterns of these genes in planta correlate well with the developmental profile of lignin deposition. Downregulation of CCR1 and caffeic acid O-methyltransferase 1 (OMT1) using an RNA interference-mediated silencing strategy caused dramatic changes in lignin level and composition in transgenic perennial ryegrass plants grown under both glasshouse and field conditions. In CCR1-deficient perennial ryegrass plants, metabolic profiling indicates the redirection of intermediates both within and beyond the core phenylpropanoid pathway. The combined results strongly support a key role for the OMT1 gene product in the biosynthesis of both syringyl- and guaiacyl-lignin subunits in perennial ryegrass. Both field-grown OMT1-deficient and CCR1-deficient perennial ryegrass plants showed enhanced digestibility without obvious detrimental effects on either plant fitness or biomass production. This highlights the potential of metabolic engineering not only to enhance the forage quality of grasses but also to produce optimal feedstock plants for biofuel production.     

Abiotic stress induces change in Cinnamoyl CoA Reductase (CCR) protein abundance and lignin deposition in developing seedlings of Leucaena leucocephala

Aboitic stress such as drought and salinity are class of major threats, which plants undergo through their lifetime. Lignin deposition is one of the responses to such abiotic stresses. The gene encoding Cinnamoyl CoA Reductase (CCR) is a key gene for lignin biosynthesis, which has been shown to be over-expressed under stress conditions. In the present study, developing seedlings of Leucaena leucocephala (Vernacular name: Subabul, White popinac) were treated with 1 % mannitol and 200 mM NaCl to mimic drought and salinity stress conditions, respectively. Enzyme linked immunosorbant assay (ELISA) based expression pattern of CCR protein was monitored coupled with Phlorogucinol/HCl activity staining of lignin in transverse sections of developing L. leucocephala seedlings under stress. Our result suggests a differential lignification pattern in developing root and stem under stress conditions. Increase in lignification was observed in mannitol treated stems and corresponding CCR protein accumulation was also higher than control and salt stress treated samples. On the contrary CCR protein was lower in NaCl treated stems and corresponding lignin deposition was also low. Developing root tissue showed a high level of CCR content and lignin deposition than stem samples under all conditions tested. Overall result suggested that lignin accumulation was not affected much in case of developing root however developing stems were significantly affected under drought and salinity stress condition.Keyword: Abiotic stress, Cinnamoyl CoA reductase, Developing seedlings, Leucaena leucocephala     

Variation in lignin and cell wall digestibility in caffeic acid O-methyltransferase down-regulated maize half-sib progenies in field experiments

Lignin content and structure were examined in seven caffeic acid O-methyltransferase antisense (COMT-AS) maize progenies and their corresponding normal inbred lines in relation to cell wall digestibility. The seven parental inbreds were chosen for their highly divergent in vitro wall digestibility. Maize plants were grown under field conditions to determine (i) if the positive effect of COMT down-regulation on wall chemistry and digestibility was similar to that previously observed for COMT-AS maize grown in the greenhouse and (ii) to what extent the genetic background was a factor in determining the effect of the transgene. All␣COMT-AS progenies displayed a significant reduction in endogenous COMT activity (14–43% residual activity). In all but one genetic background (F4), the COMT-AS gene resulted in an expected increase in wall digestibility accompanied by changes in lignin composition. These effects varied greatly among parental lines, and independently of the inherent digestibility values in the corresponding non-transformed lines. Curiously, in the highly digestible F4 background, the typical decrease in syringyl (S) unit lignin and a higher frequency of 5-OH guaiacyl lignin resulting from the introduction of the COMT-AS transgene were not observed. Our results indicate that COMT down-regulation via an antisense strategy is an efficient tool for forage maize improvement in the field.     

Genetic variations of cell wall digestibility related traits in floral stems of Arabidopsis thaliana accessions as a basis for the improvement of the feeding value in maize and forage plants

Floral stems of Arabidopsis thaliana accessions were used as a model system relative to forage plant stems in genetic variation studies of lignin content and cell wall digestibility related traits. Successive investigations were developed in a core collection of 24 Arabidopsis accessions and in a larger collection of 280 accessions. Significant genetic variation for lignin content in the cell wall, and for the two in vitro cell wall digestibility investigated traits, were found both in the core collection and in the large collection. Genotype × environment interactions, investigated in the core collection, were significant with a few genotypes contributing greatly to interactions, based on ecovalence value estimates. In the core collection, genotypes 42AV, 224AV, and 8AV had low cell wall digestibility values, whatever be the environmental conditions. Genotype 157AV, observed only in one environment, also appeared to have a low cell wall digestibility. Conversely, genotypes 236AV, 162AV, 70AV, 101AV, 83AV had high cell wall digestibility values, genotype 83AV having a slightly greater instability across differing environments than others. The well-known accession Col-0 (186AV) appeared with a medium level of cell wall digestibility and a weak to medium level of interaction between environments. The ranges of variation in cell wall digestibility traits were higher in the large collection than in the core collection of 24 accessions, these results needing confirmation due to the lower number of replicates. Accessions 295AV, 148AV, and 309AV could be models for low stem cell wall digestibility values, with variable lignin content. Similarly, accessions 83AV and 162AV, already identified from the study of the core collection, and five accessions (6AV, 20AV, 91AV, 114AV, and 223AV) could be models for high stem cell wall digestibility values. The large variations observed between Arabidopsis accessions for both lignin content and cell wall digestibility in floral stems have strengthened the use this species as a powerful tool for discovering genes involved in cell wall biosynthesis and lignification of dicotyledons forage plants. Investigations of this kind might also be applicable to monocotyledons forage plants due to the basic similarity of the genes involved in the lignin pathway of Angiosperms and the partial homology of the cell wall composition and organization of the mature vascular system in grasses and Arabidopsis.     

Functional Analysis of Metabolic Channeling and Regulation in Lignin Biosynthesis: A Computational Approach

Lignin is a polymer in secondary cell walls of plants that is known to have negative impacts on forage digestibility, pulping efficiency, and sugar release from cellulosic biomass. While targeted modifications of different lignin biosynthetic enzymes have permitted the generation of transgenic plants with desirable traits, such as improved digestibility or reduced recalcitrance to saccharification, some of the engineered plants exhibit monomer compositions that are clearly at odds with the expected outcomes when the biosynthetic pathway is perturbed. In Medicago, such discrepancies were partly reconciled by the recent finding that certain biosynthetic enzymes may be spatially organized into two independent channels for the synthesis of guaiacyl (G) and syringyl (S) lignin monomers. Nevertheless, the mechanistic details, as well as the biological function of these interactions, remain unclear. To decipher the working principles of this and similar control mechanisms, we propose and employ here a novel computational approach that permits an expedient and exhaustive assessment of hundreds of minimal designs that could arise in vivo. Interestingly, this comparative analysis not only helps distinguish two most parsimonious mechanisms of crosstalk between the two channels by formulating a targeted and readily testable hypothesis, but also suggests that the G lignin-specific channel is more important for proper functioning than the S lignin-specific channel. While the proposed strategy of analysis in this article is tightly focused on lignin synthesis, it is likely to be of similar utility in extracting unbiased information in a variety of situations, where the spatial organization of molecular components is critical for coordinating the flow of cellular information, and where initially various control designs seem equally valid.     

The pentose phosphate pathway as a source of NADPH for lignin synthesis

The aim of this work was to investigate whether the pentose phosphate pathway provides reducing power for lignin synthesis. Explants of the stem of Coleus blumei and the storage tissue of Helianthus tuberosus were cultured for 4 days on media which caused extensive lignification. [3-³H]-glucose and either [3-¹⁴C]- or [U-¹⁴C]-glucose were supplied to such 4-day-cultured explants, and also to the roots of 5-day-old seedlings of Pisum sativum. Significant amounts of ³H and ¹⁴C were recovered in syringaldehyde, vanillin, p-hydroxybenzaldehyde, and ligothio-glycollic acid from the explants of Coleus and Helianthus; and in vanillin, p-hydroxybenzaldehyde, and milled-wood lignin from pea roots. The ³H/¹⁴C ratios in these derivatives and preparations of lignin are held to indicate that much of the reducing power for lignin synthesis comes from the pentose phosphate pathway.     

Prediction of the day matter digestibility of tropical grasses using a pepsin-cellulase assay

A study was made of the accuracy of predicting dry matter digestibility in vivo from the dry matter disappearance of forage samples in pepsin (48 h) followed by Onozuka cellulase (48 h). Forty-five samples representing four tropical pasture species, Cenchrus ciliaris, Chloris gayana, Digitaria spp., Setaria spp. and one temperate grass, Lolium perenne were used.Dry matter digestibility in vivo was significantly correlated with the disappearance in pepsin-cellulase (r = 0.94) and the regression predicting dry matter digestibility in vivo had a residual standard deviation of ± 2.7 digestibility units. Separate regressions for the five grasses had RSD values ranging from ± 1.3 to ± 2.6.It was concluded that the digestibility in vivo of both tropical and temperate grasses in the range 40–76% digestibility could be accurately predicted by the pepsin-cellulase assay, provided samples of known digestibility in vivo similar to those being tested were included as standards in each run.     

Correlations among anatomical,morphological, chemical and agronomic characteristics of leaf blades in Panicum maximum genotypes

Tropical forage grasses present high growth rates and biomass yields, partly due to its C₄ photosynthetic pathway. Considering this, the anatomy of the grasses related to morphology and chemical composition of leaf blades may influence consumption and digestibility, thus interfering in the forage quality. Agronomic, morphological, anatomical and chemical characteristics of leaves of nine Panicum maximum genotypes were evaluated in Brazil, to verify if these characteristics and the associations among them may influence the quality of the leaf blades. A randomized complete blocks design was used with nine treatments and three replications. Three evaluation harvests were done in the rainy season and one in the dry season to evaluate forage yields and quality. Quality was determined through NIRS – Near Infrared Spectrometer. One day prior to each harvest, four leaf blades per plot were harvested for morphological and anatomical evaluations. Results were subjected to analysis of variance and mean comparison by Tukey test, and to simple linear and canonical correlations by SAS. Leaf width was positively correlated with mesophyll. The parenchyma bundle sheath was associated with leaf area and specific leaf area. Neutral detergent fibre was positively correlated with the parenchyma bundle sheath area and specific leaf area. The specific leaf area was negatively correlated with in vitro organic matter digestibility. Morphological differences among P. maximum genotypes did not interfere in biomass accumulation. Considering this, leaf width may be a supplementary tool, that may be used in the early phases of the process of genotype selection, for discriminating qualitatively promising high yielding materials.