The primary obstacle to producing renewable fuels from lignocellulosic biomass is a plant's recalcitrance to releasing sugars bound in the cell wall. From a sample set of wood cores representing 1,100 individual undomesticated Populus trichocarpa trees, 47 extreme phenotypes were selected across measured lignin content and ratio of syringyl and guaiacyl units (S/G ratio). This subset was tested for total sugar release through enzymatic hydrolysis alone as well as through combined hot-water pretreatment and enzymatic hydrolysis using a high-throughput screening method. The total amount of glucan and xylan released varied widely among samples, with total sugar yields of up to 92% of the theoretical maximum. A strong negative correlation between sugar release and lignin content was only found for pretreated samples with an S/G ratio < 2.0. For higher S/G ratios, sugar release was generally higher, and the negative influence of lignin was less pronounced. When examined separately, only glucose release was correlated with lignin content and S/G ratio in this manner, whereas xylose release depended on the S/G ratio alone. For enzymatic hydrolysis without pretreatment, sugar release increased significantly with decreasing lignin content below 20%, irrespective of the S/G ratio. Furthermore, certain samples featuring average lignin content and S/G ratios exhibited exceptional sugar release. These facts suggest that factors beyond lignin and S/G ratio influence recalcitrance to sugar release and point to a critical need for deeper understanding of cell-wall structure before plants can be rationally engineered for reduced recalcitrance and efficient biofuels production. L ignocellulosic biomass is the only sustainable resource in terms of cost, availability, and scale that can be converted into liquid fuels to reduce the prevailing role of petroleum in providing energy for the world's transportation needs (1, 2) and to decrease the emissions of fossil CO 2 that damage the world's climate (3). The primary obstacle to producing liquid transportation fuels by bioconversion methods is the release of sugars in high quantities at low costs from recalcitrant lignocellulosic biomass feedstocks (4, 5). Genetic modification of plants to make them less recalcitrant is a promising path to address this challenge on the feedstock side, but the effort would be greatly aided by improving understanding of the fundamental relationship between cell-wall composition and sugar release through pretreatment and enzymatic hydrolysis.In this paper, we focus on the influence of lignin content and the ratio of its syringyl and guaiacyl units (S/G ratio) on recalcitrance to sugar release, because these two traits were previously identified as dominant factors (6). Although it is generally perceived that low lignin contents increase the ability of cellulolytic enzymes to hydrolyze plant fibers (7-11), only a limited number of studies investigated the effect of lignin S/G ratio on sugar release through combined pretreatment and enzymatic hydrolysis. A...

Grass cell wall properties influence food, feed, and biofuel feedstock usage efficiency. The glucuronoarabinoxylan of grass cell walls is esterified with the phenylpropanoid-derived hydroxycinnamic acids ferulic acid (FA) and para-coumaric acid (p-CA). Feruloyl esters undergo oxidative coupling with neighboring phenylpropanoids on glucuronoarabinoxylan and lignin. Examination of rice (Oryza sativa) mutants in a grass-expanded and -diverged clade of BAHD acyl-coenzyme A-utilizing transferases identified four mutants with altered cell wall FA or p-CA contents. Here, we report on the effects of overexpressing one of these genes, OsAt10 (LOC_Os06g39390), in rice. An activation-tagged line, OsAT10-D1, shows a 60% reduction in matrix polysaccharide-bound FA and an approximately 300% increase in p-CA in young leaf tissue but no discernible phenotypic alterations in vegetative development, lignin content, or lignin composition. Two additional independent OsAt10 overexpression lines show similar changes in FA and p-CA content. Cell wall fractionation and liquid chromatography-mass spectrometry experiments isolate the cell wall alterations in the mutant to ester conjugates of a fivecarbon sugar with p-CA and FA. These results suggest that OsAT10 is a p-coumaroyl coenzyme A transferase involved in glucuronoarabinoxylan modification. Biomass from OsAT10-D1 exhibits a 20% to 40% increase in saccharification yield depending on the assay. Thus, OsAt10 is an attractive target for improving grass cell wall quality for fuel and animal feed.

Transgenic down-regulation of the Pt4CL1 gene family encoding 4-coumarate:coenzyme A ligase (4CL) has been reported as a means for reducing lignin content in cell walls and increasing overall growth rates, thereby improving feedstock quality for paper and bioethanol production. Using hybrid poplar (Populus tremula 3 Populus alba), we applied this strategy and examined field-grown transformants for both effects on wood biochemistry and tree productivity. The reductions in lignin contents obtained correlated well with 4CL RNA expression, with a sharp decrease in lignin amount being observed for RNA expression below approximately 50% of the nontransgenic control. Relatively small lignin reductions of approximately 10% were associated with reduced productivity, decreased wood syringyl/guaiacyl lignin monomer ratios, and a small increase in the level of incorporation of H-monomers (p-hydroxyphenyl) into cell walls. Transgenic events with less than approximately 50% 4CL RNA expression were characterized by patches of reddish-brown discolored wood that had approximately twice the extractive content of controls (largely complex polyphenolics). There was no evidence that substantially reduced lignin contents increased growth rates or saccharification potential. Our results suggest that the capacity for lignin reduction is limited; below a threshold, large changes in wood chemistry and plant metabolism were observed that adversely affected productivity and potential ethanol yield. They also underline the importance of field studies to obtain physiologically meaningful results and to support technology development with transgenic trees.Composed of diverse layers of cellulose microfibrils and amorphous hemicelluloses within a matrix of pectins, proteins, and lignin, the secondary cell walls of plants are diverse in their morphology, chemistry, and physiological functions. Lignification is of particular interest, as it exhibits highly predictable temporal and spatial patterning and is the last major step in the structural reinforcement of cell walls before the protoplast is dissolved (Donaldson, 2001

Summary• An association genetics approach was used to examine individual genes and alleles at the loci responsible for complex traits controlling lignocellulosic biosynthesis in black cottonwood (Populus trichocarpa). Recent interest in poplars as a source of renewable energy, combined with the vast genomic resources available, has enabled further examination of their genetic diversity.• Forty candidate genes were resequenced in a panel of 15 unrelated individuals to identify single nucleotide polymorphisms (SNPs). Eight hundred and seventy-six SNPs were successfully genotyped in a clonally replicated population (448 clones). The association population (average of 2.4 ramets per clone) was phenotyped using pyrolysis molecular beam mass spectrometry. Both single-marker and haplotype-based association tests were implemented to identify associations for composite traits representing lignin content, syringyl : guaiacyl ratio and C6 sugars.• Twenty-seven highly significant, unique, single-marker associations (false discovery rate Q < 0.10) were identified across 40 candidate genes in three composite traits. Twenty-three significant haplotypes within 11 genes were discovered in two composite traits.• Given the rapid decay of within-gene linkage disequilibrium and the high coverage of amplicons across each gene, it is likely that the numerous polymorphisms identified are in close proximity to the causative SNPs and the haplotype associations reflect information present in the associations between markers.

BackgroundThe inherent recalcitrance of woody bioenergy feedstocks is a major challenge for their use as a source of second-generation biofuel. Secondary cell walls that constitute the majority of hardwood biomass are rich in cellulose, xylan, and lignin. The interactions among these polymers prevent facile accessibility and deconstruction by enzymes and chemicals. Plant biomass that can with minimal pretreatment be degraded into sugars is required to produce renewable biofuels in a cost-effective manner.ResultsGAUT12/IRX8 is a putative glycosyltransferase proposed to be involved in secondary cell wall glucuronoxylan and/or pectin biosynthesis based on concomitant reductions of both xylan and the pectin homogalacturonan (HG) in Arabidopsis irx8 mutants. Two GAUT12 homologs exist in Populus trichocarpa, PtGAUT12.1 and PtGAUT12.2. Knockdown expression of both genes simultaneously has been shown to reduce xylan content in Populus wood. We tested the proposition that RNA interference (RNAi) downregulation of GAUT12.1 alone would lead to increased sugar release in Populus wood, that is, reduced recalcitrance, based on the hypothesis that GAUT12 synthesizes a wall structure required for deposition of xylan and that cell walls with less xylan and/or modified cell wall architecture would have reduced recalcitrance. Using an RNAi approach, we generated 11 Populus deltoides transgenic lines with 50 to 67% reduced PdGAUT12.1 transcript expression compared to wild type (WT) and vector controls. Ten of the eleven RNAi lines yielded 4 to 8% greater glucose release upon enzymatic saccharification than the controls. The PdGAUT12.1 knockdown (PdGAUT12.1-KD) lines also displayed 12 to 52% and 12 to 44% increased plant height and radial stem diameter, respectively, compared to the controls. Knockdown of PdGAUT12.1 resulted in a 25 to 47% reduction in galacturonic acid and 17 to 30% reduction in xylose without affecting total lignin content, revealing that in Populus wood as in Arabidopsis, GAUT12 affects both pectin and xylan formation. Analyses of the sugars present in sequential cell wall extracts revealed a reduction of glucuronoxylan and pectic HG and rhamnogalacturonan in extracts from PdGAUT12.1-KD lines.ConclusionsThe results show that downregulation of GAUT12.1 leads to a reduction in a population of xylan and pectin during wood formation and to reduced recalcitrance, more easily extractable cell walls, and increased growth in Populus.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0218-y) contains supplementary material, which is available to authorized users.

BackgroundAs a major component of plant cell wall, lignin plays important roles in mechanical support, water transport, and stress responses. As the main cause for the recalcitrance of plant cell wall, lignin modification has been a major task for bioenergy feedstock improvement. The study of the evolution and function of lignin biosynthesis genes thus has two-fold implications. First, the lignin biosynthesis pathway provides an excellent model to study the coordinative evolution of a biochemical pathway in plants. Second, understanding the function and evolution of lignin biosynthesis genes will guide us to develop better strategies for bioenergy feedstock improvement.ResultsWe analyzed lignin biosynthesis genes from fourteen plant species and one symbiotic fungal species. Comprehensive comparative genome analysis was carried out to study the distribution, relatedness, and family expansion of the lignin biosynthesis genes across the plant kingdom. In addition, we also analyzed the comparative synteny map between rice and sorghum to study the evolution of lignin biosynthesis genes within the Poaceae family and the chromosome evolution between the two species. Comprehensive lignin biosynthesis gene expression analysis was performed in rice, poplar and Arabidopsis. The representative data from rice indicates that different fates of gene duplications exist for lignin biosynthesis genes. In addition, we also carried out the biomass composition analysis of nine Arabidopsis mutants with both MBMS analysis and traditional wet chemistry methods. The results were analyzed together with the genomics analysis.ConclusionThe research revealed that, among the species analyzed, the complete lignin biosynthesis pathway first appeared in moss; the pathway is absent in green algae. The expansion of lignin biosynthesis gene families correlates with substrate diversity. In addition, we found that the expansion of the gene families mostly occurred after the divergence of monocots and dicots, with the exception of the C4H gene family. Gene expression analysis revealed different fates of gene duplications, largely confirming plants are tolerant to gene dosage effects. The rapid expansion of lignin biosynthesis genes indicated that the translation of transgenic lignin modification strategies from model species to bioenergy feedstock might only be successful between the closely relevant species within the same family.

We describe a high-throughput method for estimating cell-wall chemistry traits using analytical pyrolysis. The instrument used to perform the high-throughput cell-wall chemistry analysis consists of a commercially available pyrolysis unit and autosampler coupled to a custom-built molecular beam mass spectrometer. The system is capable of analyzing approximately 42 biomass samples per hour. Lignin content and syringyl to guaiacol (S/G) ratios can be estimated directly from the spectra and differences in cell wall chemistry in large groups of samples can easily be identified using multivariate statistical data analysis methods. The utility of the system is demonstrated on a set of 800 greenhouse-grown poplar trees grown under two contrasting nitrogen treatments. High-throughput analytical pyrolysis was able to determine that the lignin content varied between 13 and 28% and the S/G ratio ranged from 0.5 to 1.5. There was more cell-wall chemistry variation in the plants grown under high nitrogen conditions than trees grown under nitrogen-deficiency conditions. Analytical pyrolysis allows the user to rapidly screen large numbers of samples at low cost, using very little sample material while producing reliable and reproducible results.

Summary The genetic control of carbon allocation and partitioning in woody perennial plants is poorly understood despite its importance for carbon sequestration, biofuels and other wood‐based industries. It is also unclear how environmental cues, such as nitrogen availability, impact the genes that regulate growth, biomass allocation and wood composition in trees. We phenotyped 396 clonally replicated genotypes of an interspecific pseudo‐backcross pedigree of Populus for wood composition and biomass traits in above‐ and below‐ground organs. The loci that regulate growth, carbon allocation and partitioning under two nitrogen conditions were identified, defining the contribution of environmental cues to their genetic control. Sixty‐three quantitative trait loci were identified for the 20 traits analyzed. The majority of quantitative trait loci are specific to one of the two nitrogen treatments, demonstrating significant nitrogen‐dependent genetic control. A highly significant genetic correlation was observed between plant growth and lignin/cellulose composition, and quantitative trait loci co‐localization identified the genomic position of potential pleiotropic regulators. Pleiotropic loci linking higher growth rates to wood with less lignin are excellent targets to engineer tree germplasm improved for pulp, paper and cellulosic ethanol production. The causative genes are being identified with a genetical genomics approach.