Search

Summary Prefoldin (PFD) is a group II chaperonin that is ubiquitously present in the eukaryotic kingdom. Six subunits (PFD1‐6) form a jellyfish‐like heterohexameric PFD complex and function in protein folding and cytoskeleton organization. However, little is known about its function in plant cell wall‐related processes. Here, we report the functional characterization of a PFD gene from Populus deltoides, designated as PdPFD2.2. There are two copies of PFD2 in Populus, and PdPFD2.2 was ubiquitously expressed with high transcript abundance in the cambial region. PdPFD2.2 can physically interact with DELLA protein RGA1_8g, and its subcellular localization is affected by the interaction. In P. deltoides transgenic plants overexpressing PdPFD2.2, the lignin syringyl/guaiacyl ratio was increased, but cellulose content and crystallinity index were unchanged. In addition, the total released sugar (glucose and xylose) amounts were increased by 7.6% and 6.1%, respectively, in two transgenic lines. Transcriptomic and metabolomic analyses revealed that secondary metabolic pathways, including lignin and flavonoid biosynthesis, were affected by overexpressing PdPFD2.2. A total of eight hub transcription factors (TFs) were identified based on TF binding sites of differentially expressed genes in Populus transgenic plants overexpressing PdPFD2.2. In addition, several known cell wall‐related TFs, such as MYB3, MYB4, MYB7, TT8 and XND1, were affected by overexpression of PdPFD2.2. These results suggest that overexpression of PdPFD2.2 can reduce biomass recalcitrance and PdPFD2.2 is a promising target for genetic engineering to improve feedstock characteristics to enhance biofuel conversion and reduce the cost of lignocellulosic biofuel production.

Background Lignocellulosic biomass is recognized as a promising renewable feedstock for the production of biofuels. However, current methods for converting biomass into fermentable sugars are considered too expensive and inefficient due to the recalcitrance of the secondary cell wall. Biomass composition can be modified to create varieties that are efficiently broken down to release cell wall sugars. This study focused on identifying the key biomass components influencing plant cell wall recalcitrance that can be targeted for selection in sugarcane, an important and abundant source of biomass. Results Biomass composition and the amount of glucan converted into glucose after saccharification were measured in leaf and culm tissues from seven sugarcane genotypes varying in fiber composition after no pretreatment and dilute acid, hydrothermal and ionic liquid pretreatments. In extractives-free sugarcane leaf and culm tissue, glucan, xylan, acid-insoluble lignin (AIL) and acid-soluble lignin (ASL) ranged from 20 to 32%, 15% to 21%, 14% to 20% and 2% to 4%, respectively. The ratio of syringyl (S) to guaiacyl (G) content in the lignin ranged from 1.5 to 2.2 in the culm and from 0.65 to 1.1 in the leaf. Hydrothermal and dilute acid pretreatments predominantly reduced xylan content, while the ionic liquid (IL) pretreatment targeted AIL reduction. The amount of glucan converted into glucose after 26 h of pre-saccharification was highest after IL pretreatment (42% in culm and 63.5% in leaf) compared to the other pretreatments. Additionally, glucan conversion in leaf tissues was approximately 1.5-fold of that in culm tissues. Percent glucan conversion varied between genotypes but there was no genotype that was superior to all others across the pretreatment groups. Path analysis revealed that S/G ratio, AIL and xylan had the strongest negative associations with percent glucan conversion, while ASL and glucan content had strong positive influences. Conclusion To improve saccharification efficiency of lignocellulosic biomass, breeders should focus on reducing S/G ratio, xylan and AIL content and increasing ASL and glucan content. This will be key for the development of sugarcane varieties for bioenergy uses.

(Figure Presented) Cardoon (Cynara cardunculus) stalks were submitted to steam explosion (183°C, 5 min) with a yield of 88% and a lignin loss of around 7%. Cardoon (C) and steam exploded cardoon (CSE) were characterized by wet chemical analysis and Py-GC/MS. Total lignin and its S/G ratio were 21.6% and 1.35 in C and 22.4% and 1.40 in CSE. These samples were delignified by kraft and organosolv processes. Cardoon pulps were obtained with different yields of 45.1% for kraft and 61.2% for organosolv. Kraft pulps yields were 45.1% and 50.9%, respectively, for Ck and CSE-K, corresponding to a lignin loss of 74.7% and 72.2%. The S/G ratio was similar 1.25 and 1.23. Organosolv pulps presented higher yields (61.2% vs 73.3%) and were less delignified (30.5% vs 13.8%), and the S/G ratio was 1.54 and 1.47, respectively, for Corg and CSE-org. Overall, the condition chosen for steam explosion allowed a low mass loss but did not promote delignification and neither enhanced lignin removal during kraft and organosolv processes. © 2016 American Chemical Society.

Thioacidolysis is a method used to measure the relative content of lignin monomers bound by β‐O‐4 linkages. Current thioacidolysis methods are low‐throughput as they require tedious steps for reaction product concentration prior to analysis using standard GC methods. A quantitative thioacidolysis method that is accessible with general laboratory equipment and uses a non‐chlorinated organic solvent and is tailored for higher‐throughput analysis is reported. The method utilizes lignin arylglycerol monomer standards for calibration, requires 1–2 mg of biomass per assay and has been quantified using fast‐GC techniques including a Low Thermal Mass Modular Accelerated Column Heater (LTM MACH). Cumbersome steps, including standard purification, sample concentrating and drying have been eliminated to help aid in consecutive day‐to‐day analyses needed to sustain a high sample throughput for large screening experiments without the loss of quantitation accuracy. The method reported in this manuscript has been quantitatively validated against a commonly used thioacidolysis method and across two different research sites with three common biomass varieties to represent hardwoods, softwoods, and grasses.

Background Understanding lignin biosynthesis and composition is of central importance for sustainable bioenergy and biomaterials production. Species of the genus Miscanthus have emerged as promising bioenergy crop due to their rapid growth and modest nutrient requirements. However, lignin polymerization in Miscanthus is poorly understood. It was previously shown that plant laccases are phenol oxidases that have multiple functions in plant, one of which is the polymerization of monolignols. Herein, we link a newly discovered Miscanthus laccase, MsLAC1, to cell wall lignification. Characterization of recombinant MsLAC1 and Arabidopsis transgenic plants expressing MsLAC1 were carried out to understand the function of MsLAC1 both in vitro and in vivo. Results Using a comprehensive suite of molecular, biochemical and histochemical analyses, we show that MsLAC1 localizes to cell walls and identify Miscanthus transcription factors capable of regulating MsLAC1 expression. In addition, MsLAC1 complements the Arabidopsis lac4–2 lac17 mutant and recombinant MsLAC1 is able to oxidize monolignol in vitro. Transgenic Arabidopsis plants over-expressing MsLAC1 show higher G-lignin content, although recombinant MsLAC1 seemed to prefer sinapyl alcohol as substrate. Conclusions In summary, our results suggest that MsLAC1 is regulated by secondary cell wall MYB transcription factors and is involved in lignification of xylem fibers. This report identifies MsLAC1 as a promising breeding target in Miscanthus for biofuel and biomaterial applications.

This study focused on the structural differences of lignin after pyridine&ndash, acetic acid&ndash, water (PAW) and dioxane&ndash, acidic water (DAW) purification processes. These structural differences included the S/G ratio, condensed structure, weight-average (MW) molecular weights, &beta, O-4 linkages and sugar content. The chemical structure of the isolated crude lignin (CL), PAW purified lignin (PPL) and DAW purified lignin (DPL) was elucidated using quantitative 13C NMR, 2D-HSQC NMR spectra, thermogravimetric analysis (TGA), gel permeation chromatography (GPC) and Fourier transform infrared spectroscopy (FTIR). The results showed that the PPL fractions contain fewer condensed structures, higher S/G ratios, more &beta, O-4 linkages, higher average MW and lower thermal degradation properties compared to the CL and DPL fractions. Furthermore, the PAW process was more selective in removing condensed units and enriching S-type lignin from CL compared to the DAW process. These results provide valuable information for understanding which purification process is more suitable to be applied for lignin.

The objective of this chapter is to provide a concise overview of lignin composition and structure in different species and materials (wood, barks and nonwood plants). It includes a brief review on the lignin precursors and their polymerization as well as of the analytical tools used for lignin characterization from wet chemical to spectroscopic methods. Wood of gymnosperms is characterized by high lignin content (25–35%) and a HG-type of lignin with more guaiacyl (G) units and a small portion of p-hydroxyphenyl (H) units. Wood of angiosperms has a lignin content of 15–28%, with a GS-lignin having different proportions of syringyl (S) units. Nonwoody monocotyledon species have different lignin content (9–20%) and a HGS type of lignin, characterized by a high proportion of H units. Bark lignin content ranges from 13 to 43% and is of HGS-type with species-specific composition and different in the bark components, phloem and cork. Lignin composition and macromolecular structure are key issues to understand the properties of lignocellulosic materials and to design a lignin-based pathway within biomass biorefineries. The available information on lignin composition is still limited to a few species and plant components. This is certainly an area where more research is needed. info:eu-repo/semantics/publishedVersion

Analytical pyrolysis is used to chemically study complex molecular materials and is applied in a wide range of fields. Pyrolysis is a thermochemical process associated to the breaking of chemical bonds using thermal energy, transforming a nonvolatile compound into a volatile degradation mixture. This chapter refers to analytical pyrolysis of lignocellulosic materials, i.e., when pyrolysis is used for chemical characterization, applied to samples with small particle sizes, at 500–650°C, and with short residence times. The reactions that occur during pyrolysis of the structural components are discussed regarding the mechanisms and the pyrolysis products obtained from cellulose, hemicelluloses, and lignin. A compilation of data is made on the characterization of lignocellulosic materials using Py-GC/FID(MS) or Py-GC/MS as analytical tools including woods and barks of several species. The pyrogram profiles and important parameters on lignin chemical composition such as the H:G:S relation and the S/G ratio are summarized. Analytical pyrolysis is a versatile methodology that may be applied to characterize the lignin directly on the lignocellulosic material or after isolation from the cell wall matrix (e.g., as MWL or dioxane lignin) or from pulps or spent liquors. It is therefore an excellent tool to study lignin compositional variability in different materials and along various processing pathways. info:eu-repo/semantics/publishedVersion

Genetic analysis of wood chemical composition is often limited by the cost and throughput of direct analytical methods. The speed and low cost of Fourier transform near infrared (FT-NIR) overcomes many of these limitations, but it is an indirect method relying on calibration models that are typically developed and validated with small sample sets. In this study, we used &gt, 1500 young greenhouse grown trees from a clonally propagated single Populus family, grown at low and high nitrogen, and compared FT-NIR calibration sample sizes of 150, 250, 500 and 750 on calibration and prediction model statistics, and heritability estimates developed with pyrolysis molecular beam mass spectrometry (pyMBMS) wood chemical composition. As calibration sample size increased from 150 to 750, predictive model statistics improved slightly. Overall, stronger calibration and prediction statistics were obtained with lignin, S-lignin, S/G ratio, and m/z 144 (an ion from cellulose), than with C5 and C6 carbohydrates, and m/z 114 (an ion from xylan). Although small differences in model statistics were observed between the 250 and 500 sample calibration sets, when predicted values were used for calculating genetic control, the 500 sample set gave substantially more similar results to those obtained with the pyMBMS data. With the 500 sample calibration models, genetic correlations obtained with FT-NIR and pyMBMS methods were similar. Quantitative trait loci (QTL) analysis with pyMBMS and FT-NIR predictions identified only three common loci for lignin traits. FT-NIR identified four QTLs that were not found with pyMBMS data, and these QTLs were for the less well predicted carbohydrate traits.

Pyrolysis-gas chromatography/mass spectrometry (Py-GC-MS) was applied to measure the lignin syringyl/guaiacyl (S/G) ratio in E. dunni, E. grandis, E. nitens, E. urograndis, and E. urophylla woods. A total of 41 compounds were identified, of which 11 were derived from carbo-hydrates and 30 from lignins. The S/G ratio was calculated on the basis of the areas of peaks recorded in the pyrograms and compared with the results obtained by alkaline nitrobenzene oxidation. The values of S/G found by pyrolysis were similar for all the species using the compounds guaiacol, 4-methylguaiacol, 4-vinylguaiacol, vanillin, 4-ethylsyringol, 4-vinylsyringol, homosyringaldehyde, acetosyringone, and syringylacetone, as lignin markers. The selected markers were efficient for the deter-mination of S/G ratio in eucalyptus wood by Py-GC-MS. The Py-GC-MS technique produced results that are comparable to the nitrobenzene oxidation method, with the advantage of requiring small wood samples and a short analysis time.

E-mail address: delrio@irnase.csic.es (J.C. del Río), The lipid and lignin composition of woods from the eucalypt species Eucalyptus globulus, E. nitens, E. maidenii, E. grandis, and E. dunnii has been characterized. The lipid composition was analyzed by GC and GC/MS equipped by short- and medium-length high-temperature capillary columns which allowed the detection of intact high molecular weight compounds. Similar lipid compositions were observed in all eucalypt woods, which were dominated by sitosterol, sitosterol esters and sitosteryl 3β-D-glucopyranoside. These substance classes are mainly responsible for pitch deposition during kraft pulping of eucalypt wood. However, some quantitative differences were found in the abundances of different lipid classes, the wood from E. globulus having the lowest amounts of these pitch-forming compounds. The lignins of all eucalypt woods were analyzed “in situ” (without previous isolation) by pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS). A predominance of syringyl (S) over guaiacyl (G) lignin units was observed and the S/G ratio was in the range from 2.7 to 4.1. The E. globulus wood had the highest S/G ratio, a finding which together with its low lignin content explains its easy delignification under kraft cooking conditions.

11 pages, 5 figures, 3 tables, 18 references. Enlazamos a la presentación a congreso: http://hdl.handle.net/10261/55044., Flash-pyrolysis in the presence of tetramethylammonium hydroxide (TMAH) of woods from different species of Eucalyptus yields series of guaiacyl-type (G) and syringyl-type (S) units in slightly but characteristically different relative proportions. Such differences have been used to suggest a fine and appropriate index of the ease of delignification of the different Eucalyptus species when pulped by the Kraft process. The pyrolytic data were in agreement with those obtained from CP/MAS 13C-NMR spectra of the woods. On the other hand, the various eucalypt woods showed additional differences in their pyrolytic patterns regarding the relative amounts and structure of the high molecular weight aromatic compounds arising from the pyrolysis of polyphenolic wood constituents, which might be used as a basis for chemotaxonomic differentiation of the origin of the woods., This research has been funded by the EU under grant FAIR CT95-0560.

20 páginas.-- 7 figuras.-- 6 tablas.-- 37 referencias.-- Artículo en Prensa, Eucalypt feedstock was pretreated with Myceliophthora thermophila laccase and methyl syringate, as mediator, in a multistage sequence consisting of successive enzymatic and alkaline peroxide stages, directly on the ground wood. Lignin modification was studied by two-dimensional (2D) nuclear magnetic resonance (NMR) of wood (at the gel state) after each stage of the sequence. Decrease of guaiacyl and syringyl lignin units (with preferential removal of the former) and aliphatic (mainly β-O-4′-linked) side chains was observed along the sequence, without a substantial change in polysaccharide cross-signals along the sequence, was observed. However, the most noticeable modification was the formation of Cα-oxidized syringyl units in the enzymatic stages, which were partially removed in the alkaline extractions. Further insight was attained by 2D NMR of the cellulolytic-enzyme lignins, isolated from the pretreated samples, and the filtrates from the different stages. Additionally, a comparison of eucalypt delignification with the high redox potential laccase from Pycnoporus cinnabarinus in the presence of the synthetic mediator 1-hydroxybenzotriazole was carried out. Wood delignification already occurred at the first cycle of the pretreatment with P. cinnabarinus laccase and 1-hydroxybenzotriazole, which directly correlated with increases in glucose (~10 %) yields after enzymatic hydrolysis. In contrast, the first cycle of pretreatment with M. thermophila laccase and methyl syringate did not produce any delignification or saccharification improvement. However, at the end of the sequence, similar delignification (~50 %) of eucalypt feedstock and increases (~30 %) in glucose yields were attained in both laccase-mediator pretreatments. Finally, the influence of lignin content and the ratio of its syringyl and guaiacyl units on recalcitrance of eucalypt feedstock to enzymatic saccharification is discussed., This study was funded by the INDOX (KBBE-2013-7-613549) EU-project and the LIGNOCELL (AGL2011-25379) and HIPOP (BIO2011-2669) Spanish MICINN projects (co-financed by FEDER funds). J. Rencoret acknowledges the CSIC JAE-Doc contract of the program “Junta para la Ampliación de Estudios” co-financed by the European Social Fund (ESF). H. Lund and L. Kalum from Novozymes (Bagsvaerd, Denmark) are acknowledged for the cellulases (Celluclast and Novozym 188) and the M. thermophila laccase, Beldem (Andenne, Belgium) for the P. cinnabarinus laccase and J. Romero (ENCE, Spain) for the eucalypt wood samples. Manuel Angulo (CITIUS, University of Seville) is acknowledged for providing technical assistance in the NMR analyses.

International audience; Lignin content and composition are considered as mandatory traits of eucalyptus breeding programs, especially for pulp, paper, and bioenergy production. In this article, we used 33 Eucalyptus urophylla full-sib families of an 8 × 8 factorial design to provide estimates of genetic parameters for lignin- and growth-related traits. Secondly, from the sequencing of the 16 unrelated founders, we described the nucleotide and haplotype variability of cinnamoyl-CoA reductase (CCR), a candidate gene for lignin-related traits encoding the cinnamoyl-CoA reductase. Finally, we tested the association between CCR polymorphisms and trait variation using a mixed linear model. A high value of narrow sense heritability was obtained for lignin content (h² = 0.85) and S/G ratio (h² = 0.62) indicating that these traits are under strong genetic control. High levels of nucleotide (θπ = 0.0131) and haplotype (Hd = 0.958) diversity were detected for CCR. From an initial set of 152 biallelic single nucleotide polymorphisms (SNPs), a subset of 65 nonredundant loci was selected. Three intronic SNPs were found to be associated to the variation of S/G ratio after multiple testing correction. In the line of what has been obtained in forest trees, these SNPs explained between 2.45% and 2.87% of the genetic variance of the trait. This study demonstrates the interest of the candidate gene approach for quantitative trait nucleotide detection in Eucalyptus and paves the way to gene assisted selection of lignin composition in E. urophylla.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais In an increasingly competitive market it is necessary that the forestry companies conducting permanent search for genetic materials that provide wood with appropriate properties for a particular purpose, combined with high productivity in the shortest possible time. In this context, this research fits, since it is proposed to verify the wood properties of different genetic materials for the charcoal production, at different ages. This study evaluated three eucalyptus clones, being a Eucalyptus grandis x Eucalyptus urophylla hybrid and Eucalyptus urophylla clones at two, three, four, five and seven years old, with a 3.5 x 2.5 m spacing. The clones are from Gerdau S/A. There was effect of the age on wood properties, the three evaluated clones, providing gains in charcoal quality and increased energy production per m3, but there was no influence on the yield gravimetric charcoal. Evaluating only the wood propertie for the best genetic material selection, the GG 680 clone had the highest potential for the charcoal production. It should be noted that the three clones meet the specifications for use steel making, with satisfactory gravimetric yield. No mercado cada vez mais competitivo é necessário que as empresas florestais realizem uma busca permanente por materiais genéticos que forneçam madeira com as propriedades adequadas para uma determinada finalidade, aliada a uma elevada produtividade, em um menor tempo possível. É nesse contexto que essa pesquisa se enquadra, uma vez que se propôs a verificar as propriedades da madeira de diferentes materiais genéticos, para a produção de carvão vegetal, em diferentes idades. Neste trabalho foram avaliados três clones de eucalipto, sendo um híbrido de Eucalyptus urophylla x Eucalyptus grandis, e dois clones de Eucalyptus urophylla, nas idades de três, quatro, cinco e sete anos, com espaçamento de 3,5 x 2,5m. Os clones são provenientes da Gerdau S/A. Verifica-se que houve efeito da idade nas propriedades da madeira, dos três clones avaliados, proporcionando ganhos na qualidade do carvão e aumento na produção de energia por m3, mas não houve influência no rendimento gravimétrico em carvão vegetal. Avaliando somente as propriedades da madeira para a seleção do melhor material genético, o clone GG 680 apresentou o maior potencial para a produção de carvão vegetal. Deve-se ressaltar que os três clones atendem as especificações para uso siderúrgico, com rendimento gravimétrico satisfatório.

The use of analytical pyrolysis combined with gas chromatography/mass spectrometry (Py-GC/MS) to determine the syringyl/guaiacyl ratio (S/G) in lignins from Eucalyptus spp woods was investigated. Sample of E. grandis and "E. urograndis" wood, with and without extractives, were subjected to pyrolysis from 300 oC to 600 oC. The products that results from pyrolysis were identified by mass spectrometry and the S/G ratio was determined based on the areas of the peaks corresponding to the guaiacyl and syringyl derivatives. The best S/G estimation is achieved when pyrolysis is carried out at 550 oC. Extractives and carbohydrate present in the woods do not interfere with the results.

The use of analytical pyrolysis combined with gas chromatography/mass spectrometry (Py-GC/MS) to determine the syringyl/guaiacyl ratio (S/G) in lignins from Eucalyptus spp woods was investigated. Sample of E. grandis and "E. urograndis" wood, with and without extractives, were subjected to pyrolysis from 300 ºC to 600 ºC. The products that results from pyrolysis were identified by mass spectrometry and the S/G ratio was determined based on the areas of the peaks corresponding to the guaiacyl and syringyl derivatives. The best S/G estimation is achieved when pyrolysis is carried out at 550 ºC. Extractives and carbohydrate present in the woods do not interfere with the results.

1)Instituto de Recursos Naturales y Agrobiología de Sevilla, P.O. Box 1052, 41080-Seville, Spain; 2)ENCE-CIT, Ctra. Campañó, Ribeiro-Vao, 36157 Pontevedra, Spain; 3)Centro de Investigaciones Biológicas, Ramiro de Maeztu 9, E-28040 Madrid, Spain. E-mail address: delrio@irnase.csic.es (J.C. del Río), A set of Eucalyptus globulus woods from different origins and growth conditions, giving a wide range of pulp yields (from 40% to 60%) upon kraft cooking, was analyzed using Py-GC/MS. The carbohydrate-derived compounds and the compounds derived from syringyl (S) and guaiacyl (G) lignin units were analyzed and the lignin/carbohydrate and S/G ratios determined. Eucalypt woods giving higher pulp yield released higher amounts of S-type compounds upon Py-GC/MS. A significant correlation was observed between pulp yield and the lignin composition in terms of the S/G ratio. By contrast, the lignin/carbohydrate ratio obtained by Py-GC/MS, did not show a significant correlation with the pulp yield. This indicates that the lignin composition is a more important parameter influencing pulp yield than the lignin content., This study has been supported by Grupo Empresarial ENCE S.A. and the Spanish Ministerio de Ciencia y Tecnología (MCyT) (project AGL2002-00393). A.G. acknowledges a "Ramón y Cajal" contract of the Spanish MCyT.