Polymer properties of softwood organosolv lignins produced in two different reactor systems

Lignin, the second most abundant biopolymer on earth and with a predominantly aromatic structure, has the potential to be a raw material for valuable chemicals and other bio-based chemicals. In industry, lignin is underutilized by being used mostly as a fuel for producing thermal energy. Valorization of lignin requires knowledge of the structure and different linkages in the isolated lignin, making the study of structure of lignin important. In this article, lignin samples isolated from two types of reactors (autoclave reactor and displacement reactor) were analyzed by FT-IR, size exclusion chromatography, thermogravimetric analysis (TGA), and Py-GC-MS. The average molecular mass of the organosolv lignins isolated from the autoclave reactor decreased at higher severities, and FT-IR showed an increase in free phenolic content with increasing severity. Except for molecular mass and molecular mass dispersity, there were only minor differences between lignins isolated from the autoclave reactor and lignins isolated from the displacement reactor. Carbohydrate analysis, Py-GC–MS and TGA showed that the lignin isolated using either of the reactor systems is of high purity, suggesting that organosolv lignin is a good candidate for valorization.     

THESEUS1 is involved in tunicamycin-induced root growth inhibition,ectopic lignin deposition,and cell wall damage-induced unfolded protein response

Endoplasmic reticulum (ER) stress activates unfolded protein responses (UPRs), such as promoting protein folding under the control of specific gene expression. Our previous study showed that ER stress induced by ER stress inducers such as tunicamycin (Tm), an inhibitor of N-linked glycan synthesis, causes ectopic lignin deposition in Arabidopsis roots, but the relationship between UPR and ectopic lignin deposition remains unclear. The receptor-like kinase THESEUS1 (THE1) has been shown to sense cell wall damage (CWD) induced in Arabidopsis by cellulose synthase inhibitors such as isoxaben (ISO) and to activate ectopic lignin deposition. In this study, we assessed the involvement of THE1 in ectopic lignin deposition caused by the ER stress inducer Tm. The loss-of-function mutation of THE1, the1-3, suppressed Tm-induced root growth inhibition and ectopic lignin deposition, revealing that THE1 is involved in root growth defects and ectopic lignin deposition caused by ER stress. Similarly, ISO treatment induced ectopic lignin deposition as well as the expression of the UPR marker genes binding protein 3 (BiP3) and ER-localized DnaJ 3b (ERdj3b). Conversely, in the the1-3 mutant, ISO-induced ectopic lignin deposition and the expression of BiP3 and ERdj3b were suppressed. These results showed that THE1 is involved in not only root growth inhibition and ectopic lignin deposition caused by ER stress but also CWD-induced UPR.     

Relationship between hydroxycinnamic acid content,lignin composition and digestibility of maize silages in sheep

Cell wall-bound hydroxycinnamic acids and the composition of lignin were studied in relation to the digestibility of a collection of 91 maize silages in wethers. Total lignin and guaiacyl content showed the highest correlation coefficients with digestibility. Using the above-mentioned chemical parameters, eight equations were also developed to predict digestibility. The prediction of organic matter digestibility produced a high adjusted R ² value (0.487) using total lignin, guaiacyl, esterified ferulic acid and esterified p-coumaric acid content as predictors. The prediction of in vivo dry matter digestibility produced a higher adjusted R ² value (0.516) using the same variables as predictors. Cell wall digestibility depends on a multiplicity of factors and it is not possible to attribute a causal effect on in vivo digestibility to any single factor. However, total lignin, guaiacyl and p-coumaric acid content emerge as good predictors of digestibility.     

Combined histochemistry and autofluorescence for identifying lignin distribution in cell walls

Histological staining methods commonly used for detecting cellulose and lignin in cell walls were combined with epifluorescence microscopy to visualize differences in lignification between and within cellular elements. We tested our approach on sections of one-year-old branches of Fraxinus ornus L., Myrtus communis L., Olea europaea L., Pistacia lentiscus L. and Rhamnus alaternus L., containing both normal and tension wood. Sections were subjected to various staining techniques, viz. safranin O, safranin O/fast green FCF, and alcoholic solutions of safranin O/astra blue, according to the commonly accepted protocols. Stained and unstained sections were compared using both light and epifluorescence microscopy. Safranin O with or without counterstaining hid the strong fluorescence of vessel walls, cell corners and middle lamellae allowing the secondary wall fibers to fluoresce more clearly. Epifluorescence microscopy applied to stained sections showed more cell wall details than autofluorescence of unstained sections or white light microscopy of counterstained sections. This simple approach proved reliable and valuable for detecting differences in lignification in thick sections without the need for costly equipment.     

Combined histochemistry and autofluorescence for identifying lignin distribution in cell walls

Histological staining methods commonly used for detecting cellulose and lignin in cell walls were combined with epifluorescence microscopy to visualize differences in lignification between and within cellular elements. We tested our approach on sections of one-year-old branches of Fraxinus ornus L., Myrtus communis L., Olea europaea L., Pistacia lentiscus L. and Rhamnus alaternus L., containing both normal and tension wood. Sections were subjected to various staining techniques, viz. safranin O, safranin O/fast green FCF, and alcoholic solutions of safranin O/astra blue, according to the commonly accepted protocols. Stained and unstained sections were compared using both light and epifluorescence microscopy. Safranin O with or without counterstaining hid the strong fluorescence of vessel walls, cell corners and middle lamellae allowing the secondary wall fibers to fluoresce more clearly. Epifluorescence microscopy applied to stained sections showed more cell wall details than autofluorescence of unstained sections or white light microscopy of counterstained sections. This simple approach proved reliable and valuable for detecting differences in lignification in thick sections without the need for costly equipment.     

Stem wood properties of Populus tremuloides, Betula papyrifera and Acer saccharum saplings after 3 years of treatments to elevated carbon dioxide and ozone

The aim of this study was to examine the effects of elevated carbon dioxide [CO₂] and ozone [O₃] and their interaction on wood chemistry and anatomy of five clones of 3‐year‐old trembling aspen (Populus tremuloides Michx.). Wood chemistry was studied also on paper birch (Betula papyrifera Marsh.) and sugar maple (Acer saccharum Marsh.) seedling‐origin saplings of the same age. Material for the study was collected from the Aspen Free‐Air CO₂ Enrichment (FACE) experiment in Rhinelander, WI, USA, where the saplings had been exposed to four treatments: control (C; ambient CO₂, ambient O₃), elevated CO₂ (560 ppm during daylight hours), elevated O₃ (1.5 × ambient during daylight hours) and their combination (CO₂+O₃) for three growing seasons (1998–2000). Wood chemistry responses to the elevated CO₂ and O₃ treatments differed between species. Aspen was most responsive, while maple was the least responsive of the three tree species. Aspen genotype affected the responses of wood chemistry and, to some extent, wood structure to the treatments. The lignin concentration increased under elevated O₃ in four clones of aspen and in birch. However, elevated CO₂ ameliorated the effect. In two aspen clones, nitrogen in wood samples decreased under combined exposure to CO₂ and O₃. Soluble sugar concentration in one aspen clone and starch concentration in two clones were increased by elevated CO₂. In aspen wood, α‐cellulose concentration changed under elevated CO₂, decreasing under ambient O₃ and slightly increasing under elevated O₃. Hemicellulose concentration in birch was decreased by elevated CO₂ and increased by elevated O₃. In aspen, elevated O₃ induced statistically significant reductions in distance from the pith to the bark and vessel lumen diameter, as well as increased wall thickness and wall percentage, and in one clone, decreased fibre lumen diameter. Our results show that juvenile wood properties of broadleaves, depending on species and genotype, were altered by atmospheric gas concentrations predicted for the year 2050 and that CO₂ ameliorates some adverse effects of elevated O₃ on wood chemistry.     

Over-expression of F5H in COMT-deficient Arabidopsis leads to enrichment of an unusual lignin and disruption of pollen wall formation

The presence of the phenylpropanoid polymer lignin in plant cell walls impedes breakdown of polysaccharides to the fermentable sugars that are used in biofuel production. Genetically modified plants with altered lignin properties hold great promise to improve biomass degradability. Here, we describe the generation of a new type of lignin enriched in 5-hydroxy-guaiacyl units by over-expressing ferulate 5-hydroxylase in a line of Arabidopsis lacking caffeic acid O-methyltransferase. The lignin modification strategy had a profound impact on plant growth and development and cell-wall properties, and resulted in male sterility due to complete disruption of formation of the pollen wall. The modified plants showed significantly improved cell-wall enzymatic saccharification efficiency without a reduction in post-harvest biomass yield despite the alterations in the overall growth morphology. This study demonstrated the plasticity of lignin polymerization in terms of incorporation of unusual monomers that chemically resemble conventional monomers, and also revealed the link between the biosynthetic pathways of lignin and the pollen wall-forming sporopollenin.     

Vitrification of carnation in vitro: Changes in cell wall mechanical properties,cellulose and lignin content

Vitrification of internodes of carnation was brought about by culturing in liquid medium. Cell wall extensibility of these internodes was kinetically followed in comparison to that of normal plants using the constant stress method. Liquid culture induced increased immediate and total deformation capacities of the walls from the second day. Measurements indicated that these deformation capacities involved plastic properties rather than elastic ones. These changes were paralleled by decreased relative levels of cellulose and lignin.     

Diferulic and ferulic acid in the cell wall of Avena coleoptiles—Their relationships to mechanical properties of the cell wall

Alkaline hydrolysis liberated ferulic and diferulic acid from polysaccharides of the Avena coleoptile ( Avena sativa L. cv. Victory I) cell walls. The amount of the two phenolic acids bound to cell walls increased substantially at day 4–5 after sowing, when the growth rate of the coleoptile started to decrease. The level of these acids was almost constant from the tip to base in 3-day-old coleoptiles, but increased toward the basal zone in 4- and 5-day-old ones. The ratio of diferulic acid to ferulic acid was almost constant irrespective of coleoptile age and zone. An increase in the amount of ferulic and diferulic acids bound to cell wall polysaccharides correlated with a decrease in extensibility and with an increase in minimum stress-relaxation time and relaxation rate of the cell wall. The level of lignin in the cellulose fraction increased as coleoptiles aged, but this increase did not correlate with changes in mechanical properties of the cell walls. These results suggest that ferulic acid, ester-linked to cell wall polysaccharides, is oxidized to give diferulic acid, which makes the cell wall mechanically rigid by cross-linking matrix polysaccharides and results in limited cell extension growth. In addition, it is probable that the step of feruloylation of cell wall polysaccharides is rate-limiting in the formation of in-termolecular bridges by diferulic acid in Avena coleoptile cell walls.     

Evolution of coumaroyl conjugate 3‐hydroxylases in land plants: lignin biosynthesis and defense

Multiple adaptations were necessary when plants conquered the land. Among them were soluble phenylpropanoids related to plant protection and lignin necessary for upright growth and long‐distance water transport. Cytochrome P450 monooxygenase 98 (CYP98) catalyzes a rate‐limiting step in phenylpropanoid biosynthesis. Phylogenetic reconstructions suggest that a single copy of CYP98 founded each major land plant lineage (bryophytes, lycophytes, monilophytes, gymnosperms and angiosperms), and was maintained as a single copy in all lineages but the angiosperms. In angiosperms, a series of independent gene duplications and losses occurred. Biochemical assays in four angiosperm species tested showed that 4‐coumaroyl‐shikimate, a known intermediate in lignin biosynthesis, was the preferred substrate of one member in each species, while independent duplicates in Populus trichocarpa and Amborella trichopoda each showed broad substrate ranges, accepting numerous 4‐coumaroyl‐esters and ‐amines, and were thus capable of producing a wide range of hydroxycinnamoyl conjugates. The gymnosperm CYP98 from Pinus taeda showed a broad substrate range, but preferred 4‐coumaroyl‐shikimate as its best substrate. In contrast, CYP98s from the lycophyte Selaginella moellendorffii and the fern Pteris vittata converted 4‐coumaroyl‐shikimate poorly in vitro, but were able to use alternative substrates, in particular 4‐coumaroyl‐anthranilate. Thus, caffeoyl‐shikimate appears unlikely to be an intermediate in monolignol biosynthesis in non‐seed vascular plants, including ferns. The best substrate for CYP98A34 from the moss Physcomitrella patens was also 4‐coumaroyl‐anthranilate, while 4‐coumaroyl‐shikimate was converted to lower extents. Despite having in vitro activity with 4‐coumaroyl‐shikimate, CYP98A34 was unable to complement the Arabidopsis thaliana cyp98a3 loss‐of‐function phenotype, suggesting distinct properties also in vivo.     

The pattern of distribution of pectin, peroxidase and lignin in the middle lamella of secondary xylem fibres in alfalfa (Medicago sativa)

BACKGROUNDS AND AIMS: Information on the micro-distribution of lignin within the middle lamella is only just beginning to emerge. This paper provides evidence of marked heterogeneity in the micro-distribution of lignin, pectin, peroxidase and hydrogen peroxide in the middle lamella of alfalfa (Medicago sativa). METHODS: Specimens from alfalfa stems were collected and processed for transmission electron microscopy. The middle lamella architecture was examined prior to and during lignification, using transmission electron microscopy in combination with pectin- and lignin-specific staining. In addition, immuno-gold labelling of peroxidase and cytochemical localization of hydrogen peroxide (H2O2) were undertaken. KEY RESULTS: Lignin showed inhomogeneity in its distribution in the middle lamella. It was found that the distribution of pectin was irregular and corresponded to the pattern of deposited lignin. Additionally, a similarity in the pattern of the deposited lignin to the pattern of distribution of peroxidase and H2O2 was also observed. CONCLUSIONS: Irregular distribution of pectin in the middle lamella may be related to subsequent inhomegeneity in lignin in this region.     

Protein charge determination and implications for interactions in cell extracts

Decades of dilute‐solution studies have revealed the influence of charged residues on protein stability, solubility and stickiness. Similar characterizations are now required in physiological solutions to understand the effect of charge on protein behavior under native conditions. Toward this end, we used free boundary and native gel electrophoresis to explore the charge of cytochrome c in buffer and in Escherichia coli extracts. We find that the charge of cytochrome c was ～2‐fold lower than predicted from primary structure analysis. Cytochrome c charge was tuned by sulfate binding and was rendered anionic in E. coli extracts due to interactions with macroanions. Mutants in which three or four cationic residues were replaced with glutamate were charge‐neutral and “inert” in extracts. A comparison of the interaction propensities of cytochrome c and the mutants emphasizes the role of negative charge in stabilizing physiological environments. Charge–charge repulsion and preferential hydration appear to prevent aggregation. The implications for molecular organization in vivo are discussed.     

天童山阔叶木本植物叶片大小与叶脉密度及单位叶脉长度细胞壁干质量的关系

叶片大小是植物生态策略中的一个关键性状, 而叶脉是叶内主要的支撑和输导结构, 对叶片的生长发育具有重要的影响。该研究以天童山38种阔叶木本植物为研究对象, 以叶片面积、干质量和周长表征叶片大小, 采用标准化主轴估计(SMA)方法和系统发育独立比较(PIC)分析主脉密度、细脉密度和总叶脉密度, 以及各级叶脉单位长度的细胞壁干质量与叶片大小之间的关系, 拟从叶片内部结构和资源分配策略的角度探明叶片大小与叶脉结构之间的变化关系及生态学意义。研究结果显示: (1)叶片大小与主脉密度极显著负相关, 细脉密度以及总叶脉密度与叶片大小关系不显著, 表明叶片越小, 主脉密度越高, 而细脉密度与叶片大小无关; (2)单位主脉长度的细胞壁干质量与叶片大小极显著正相关, 单位细脉和总叶脉长度的细胞壁干质量与叶片大小的相关性均不显著, 表明随着叶片的增大, 单位主脉长度的细胞壁干质量显著增加, 而细脉的细胞壁干质量与叶片大小无关; (3)主脉密度与单位主脉长度的细胞壁干质量之间是斜率显著大于-1的负异速生长关系, 表明主脉密度随单位主脉长度的细胞壁干质量增加而显著下降, 两者之间存在权衡关系, 而单位细脉长度的细胞壁干质量与细脉密度关系不显著。上述结果表明, 与大叶片相比, 小叶中通常具有较高的主脉密度, 这不仅是叶片发育过程中叶形变化调控的结果, 也是单位叶脉长度的细胞壁干质量调控的结果, 单位叶脉长度的细胞壁干质量是导致叶片大小与主、细脉密度之间不同变化关系的直接因素。该研究结果为我们理解全球范围内叶片大小变化的生物地理分布模式以及植物对环境的适应策略提供了参考。     

Genotype,development and tissue-derived variation of cell-wall properties in the lignocellulosic energy crop Miscanthus

Background and Aims

Species and hybrids of the genus Miscanthus contain attributes that make them front-runners among current selections of dedicated bioenergy crops. A key trait for plant biomass conversion to biofuels and biomaterials is cell-wall quality; however, knowledge of cell-wall composition and biology in Miscanthus species is limited. This study presents data on cell-wall compositional changes as a function of development and tissue type across selected genotypes, and considers implications for the development of miscanthus as a sustainable and renewable bioenergy feedstock.

Methods

Cell-wall biomass was analysed for 25 genotypes, considering different developmental stages and stem vs. leaf compositional variability, by Fourier transform mid-infrared spectroscopy and lignin determination. In addition, a Clostridium phytofermentans bioassay was used to assess cell-wall digestibility and conversion to ethanol.

Key Results

Important cell-wall compositional differences between miscanthus stem and leaf samples were found to be predominantly associated with structural carbohydrates. Lignin content increased as plants matured and was higher in stem tissues. Although stem lignin concentration correlated inversely with ethanol production, no such correlation was observed for leaves. Leaf tissue contributed significantly to total above-ground biomass at all stages, although the extent of this contribution was genotype-dependent.

Conclusions

It is hypothesized that divergent carbohydrate compositions and modifications in stem and leaf tissues are major determinants for observed differences in cell-wall quality. The findings indicate that improvement of lignocellulosic feedstocks should encompass tissue-dependent variation as it affects amenability to biological conversion. For gene–trait associations relating to cell-wall quality, the data support the separate examination of leaf and stem composition, as tissue-specific traits may be masked by considering only total above-ground biomass samples, and sample variability could be mostly due to varying tissue contributions to total biomass.     

Reactivity of bacterial and fungal laccases with lignin under alkaline conditions

The ability of Streptomyces ipomoea laccase to polymerize secoisolariciresinol lignan and technical lignins was assessed. The reactivity of S. ipomoea laccase was also compared to that of low redox fungal laccase from Melanocarpus albomyces using low molecular mass p-coumaric, ferulic and sinapic acid as well as natural (acetosyringone) and synthetic 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) mediators as substrates. Oxygen consumption measurement, MALDI-TOF MS and SEC were used to follow the enzymatic reactions at pH 7, 8, 9 and 10 at 30 °C and 50 °C. Polymerization of lignins and lignan by S. ipomoea laccase under alkaline reaction conditions was observed, and was enhanced in the presence of acetosyringone almost to the level obtained with M. albomyces laccase without mediator. Reactivities of the enzymes towards acetosyringone and TEMPO were similar, suggesting exploitation of the compounds and low redox laccase in lignin valorization under alkaline conditions. The results have scientific impact on basic research of laccases.     

The interaction between residues 62 and 193 play a key role in activity and structural stability of arginine kinase

The purpose of this study is to clarify that the amino acid residues (Asp62 and Arg193) are responsible for the activity and stability of arginine kinase (AK). The amino acid residues Asp62 (D62) and Arg193 (R193) are strictly conserved in monomeric AKs and form an ion pair in the transition state analogue complex. In this research, we replaced D62 with glutamate (E) or glycine (G) and R193 with lysine (K) or glycine (G). The mutants of D62E and R193K retained almost 90% of the wild-type activity, whereas D62G and R193G had a pronounced loss in activity. A detailed comparison was made between the physic-chemical properties and conformational changes of wild-type AK and the mutants by means of ultraviolet (UV) difference and fluorescence spectra. The results indicated that the conformation of all of the mutants had been changed and the stability in a urea solution was also reduced. We speculated that the hydrogen bond and electrostatic interactions formed between residues 62 and 193 play a key role in stabilizing the structure and mediating the synergism in substrate binding of arginine kinase from greasyback shrimp (Metapenaeus ensis).