Acetyl Bromide Soluble Lignin Research Articles

Unveiling the compositional remodelling of Arbutus unedo L. fruits during ripening

• The architecture of the cell wall of strawberry tree ( Arbutus unedo L.) fruits, during four ripening stages, is herein elucidated for the first time. • Glucose and xylose are the major neutral sugars in the cell wall, similarly to other Ericaceae. • A. unedo fruits are unusually rich in lignin. • Multiple structures of aggregated sclereids are observed in the fruits, thus unveiling their peculiar gritty texture. • Sclereids are the source of high levels of xylose and lignin. Arbutus unedo is a Mediterranean shrub or tree that produces edible fruits with promising applications for the food industry and nutraceutical applications. A deep understanding of the cell wall structure and modifications of its components during fruit softening would benefit the development of new food products and retail of fresh fruits. Aiming to enhance the valorization of A. unedo fruits, its cell wall was characterized, for the first time, during ripening. In this study, four distinct ripening stages (green, yellow, orange and red) were considered. Several preliminary quality parameters were determined. The nature of the compounds making part of the cell walls were elucidated through Fourier‑transform infrared spectroscopy in attenuated total reflectance mode (FTIR-ATR) and spectral datasets were analyzed using unsupervised principal component analysis (PCA). High-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was employed to assess monosaccharide composition, and lignin measurements were performed following the acetyl bromide soluble lignin methodology. In addition, scanning electron microscopy (SEM) analysis allowed to observe some relevant aspects of the microstructure of fruits. Significant modifications on the composition of these fruits were mostly found by the end of the ripening process when the fruits reached the red-ripe stage. Glucose, xylose and arabinose were determined as the major neutral cell wall sugars. Furthermore, the microscopy assessment unveiled the abundance of aggregated sclereids, and lignin measurements showed that A. unedo fruits are highly lignified. The richness in lignin and monosaccharides, such as glucose and xylose, may suggest the role displayed by that polyphenol and hemicelluloses like xylans and xyloglucans, which is supported by FTIR-ATR spectral assignments. It is hypothesised that the high amounts of xylose and lignin herein determined are highly associated with the sclereids. Relevant data were also obtained for the fundamental understanding of the ripening of other Ericaceae fruits and other berries.

Variability for cell-wall and yield components in commercial sugarcane (Saccharum spp.) progeny: contrasts with parental lines and energy cane

ABSTRACT Sugarcane (Saccharum spp.) is a genetically complex crop with great potential for the second-generation (2 G) ethanol industry. Despite this, there is scarce knowledge of the variability of bagasse cell-wall components and its association with agronomic traits that could be used in the selection of cultivars with improved bagasse digestibility. In this work, the acid detergent fiber method was used to determine cellulose, hemicellulose, and acid detergent lignin (ADL) in a sugarcane progeny from crossing two commercial cultivars, and in two energy-cane biotypes. Additionally, acetyl bromide-soluble lignin was determined and compared with ADL values. Despite the crossed parents showing similar bagasse composition, transgressive inheritance observed in the progeny for all bagasse cell-wall components underlines the possibility of improving sugarcane for specific bioenergetic uses. Additionally, the low association between cell-wall and yield components found in this work, suggests that indirect selection of cell-wall components for 2 G ethanol industry through agronomic traits would have a limited impact on improving the biomass composition.

Lignin Quantification of Papyri by TGA-Not a Good Idea.

Papyri belong to the oldest writing grounds in history. Their conservation is of the highest importance in preserving our cultural heritage, which is best achieved based on an extensive knowledge of the materials’ constituents to choose a tailored conservation approach. Thermogravimetric Analysis (TGA) has been widely employed to quantify cellulose and lignin in papyrus sheets, yielding reported lignin contents of 25% to 40%. In this work, the TGA method conventionally used for papyrus samples was repeated and compared to other lignin determination approaches (Klason-lignin and acetyl bromide-soluble lignin). TGA can lead to a large overestimation of the lignin content of commercial papyrus sheets (~27%) compared to the other methods (~5%). A similar overestimation of the lignin content was found for the pith and rind of the native papyrus plant. We concluded that the TGA method should, therefore, not be used for lignin quantification.

Insights Into the Mechanisms Implicated in Pinus pinaster Resistance to Pinewood Nematode.

Pine wilt disease (PWD), caused by the plant–parasitic nematode Bursaphelenchus xylophilus, has become a severe environmental problem in the Iberian Peninsula with devastating effects in Pinus pinaster forests. Despite the high levels of this species' susceptibility, previous studies reported heritable resistance in P. pinaster trees. Understanding the basis of this resistance can be of extreme relevance for future programs aiming at reducing the disease impact on P. pinaster forests. In this study, we highlighted the mechanisms possibly involved in P. pinaster resistance to PWD, by comparing the transcriptional changes between resistant and susceptible plants after infection. Our analysis revealed a higher number of differentially expressed genes (DEGs) in resistant plants (1,916) when compared with susceptible plants (1,226). Resistance to PWN is mediated by the induction of the jasmonic acid (JA) defense pathway, secondary metabolism pathways, lignin synthesis, oxidative stress response genes, and resistance genes. Quantification of the acetyl bromide-soluble lignin confirmed a significant increase of cell wall lignification of stem tissues around the inoculation zone in resistant plants. In addition to less lignified cell walls, susceptibility to the pine wood nematode seems associated with the activation of the salicylic acid (SA) defense pathway at 72 hpi, as revealed by the higher SA levels in the tissues of susceptible plants. Cell wall reinforcement and hormone signaling mechanisms seem therefore essential for a resistance response.

PRX2 and PRX25, peroxidases regulated by COG1, are involved in seed longevity in Arabidopsis.

Permeability is a crucial trait that affects seed longevity and is regulated by different polymers including proanthocyanidins, suberin, cutin and lignin located in the seed coat. By testing mutants in suberin transport and biosynthesis, we demonstrate the importance of this biopolymer to cope with seed deterioration. Transcriptomic analysis of cog1-2D, a gain-of-function mutant with increased seed longevity, revealed the upregulation of several peroxidase genes. Reverse genetics analysing seed longevity uncovered redundancy within the seed coat peroxidase gene family; however, after controlled deterioration treatment, seeds from the prx2 prx25 double and prx2 prx25 prx71 triple mutant plants presented lower germination than wild-type plants. Transmission electron microscopy analysis of the seed coat of these mutants showed a thinner palisade layer, but no changes were observed in proanthocyanidin accumulation or in the cuticle layer. Spectrophotometric quantification of acetyl bromide-soluble lignin components indicated changes in the amount of total polyphenolics derived from suberin and/or lignin in the mutant seeds. Finally, the increased seed coat permeability to tetrazolium salts observed in the prx2 prx25 and prx2 prx25 prx71 mutant lines suggested that the lower permeability of the seed coats caused by altered polyphenolics is likely to be the main reason explaining their reduced seed longevity.

Relationships between Cell Wall Digestibility and Lignin Content as Influenced by Lignin Type and Analysis Method

Difficulties in measuring the lignin content of forage and biomass crops confounds efforts by plant breeders and molecular biologists to grasp how shifts in lignin chemistry affect fiber digestibility. In this study, relationships between digestibility and lignin content were examined by artificially lignifying Zea mays L. cell walls with bulk polymers composed of p‐hydroxyphenyl‐guaiacyl (HG) and guaiacyl (G) units or with endwise polymers composed of G, guaiacyl‐syringyl (GS), and guaiacyl‐syringyl‐p‐coumaric acid (GSpCA) units. Regressions of in vitro rumen gas production or nonfermented polysaccharides (NFP) vs. lignin content accurately measured by an indirect spectrophotometric method revealed that G‐bulk and especially syringyl (S)‐rich endwise polymers were the least inhibitory to digestion. While providing biased estimates at low lignin contents, the acetyl bromide‐soluble lignin (ABSL) assay correctly ranked how endwise polymers affected the digestibility of highly lignified cell walls, but the method required unique absorption coefficients to assess relationships for bulk polymers. Conversely, the Klason lignin (KL) method provided more accurate estimates at low lignin contents, but varying recovery of KL among lignin types created bias for highly lignified cell walls. Overall, our results indicate that S‐rich lignins are the least inhibitory to digestion, but detecting desirable shifts in lignin chemistry will require the judicious use of several analytical methods.

Characterization of Miscanthus cell wall polymers.

Efficient utilization of lignocellulosic Miscanthus biomass for the production of biochemicals, such as ethanol, is challenging due to its recalcitrance, which is influenced by the individual plant cell wall polymers and their interactions. Lignocellulosic biomass composition differs depending on several factors, such as plant age, harvest date, organ type, and genotype. Here, four selected Miscanthus genotypes (Miscanthus sinensis, Miscanthus sacchariflorus, Miscanthus × giganteus, Miscanthus sinensis × Miscanthus sacchariflorus hybrid) were grown and harvested, separated into stems and leaves, and characterized for their non‐starch polysaccharide composition and structures, lignin contents and structures, and hydroxycinnamate profiles (monomers and ferulic acid dehydrodimers). Polysaccharides of all genotypes are mainly composed of cellulose and low‐substituted arabinoxylans. Ratios of hemicelluloses to cellulose were comparable, with the exception of Miscanthus sinensis that showed a higher hemicellulose/cellulose ratio. Lignin contents of Miscanthus stems were higher than those of Miscanthus leaves. Considering the same organs, the four genotypes did not differ in their Klason lignin contents, but Miscanthus × giganteus showed the highest acetylbromide soluble lignin content. Lignin polymers isolated from stems varied in their S/G ratios and linkage type distributions across genotypes. p‐Coumaric acid was the most abundant ester‐bound hydroxycinnamte monomer in all samples. Ferulic acid dehydrodimers were analyzed as cell wall cross‐links, with 8‐5‐coupled diferulic acid being the main dimer, followed by 8‐O‐4‐, and 5‐5‐diferulic acid. Contents of p‐coumaric acid, ferulic acid, and ferulic acid dimers varied depending on genotype and organ type. The largest amount of cell wall cross‐links was analyzed for Miscanthus sinensis.

Maturation-related changes of carrot lignins.

Lignified cell walls are important factors for textural and physiological properties of plant-based foods. However, carrot lignins and their modifications during maturation are poorly described. The objective of this study was to describe carrot lignins in detail and to study lignin structural alterations at later stages of maturity. Klason and acetyl bromide soluble lignin contents of insoluble fibers of carrots harvested at different times (26, 29 and 35 weeks after seeding) ranged between 46.38 and 62.68 g kg-1 and between 19.79 and 28.08 g kg-1 , respectively. As determined by both 2D-nuclear magnetic resonance and the derivatization followed by reductive cleavage method, coniferyl alcohol heavily dominated the traditional monolignol composition in carrot lignins, independently of harvest times. By using 2D-nuclear magnetic resonance experiments on isolated lignins, p-hydroxybenzoate was identified as a less common lignin constituent, attached to lignin γ-hydroxyl groups and being increasingly incorporated with maturation. β-Aryl ethers, phenylcoumaran, resinol and dibenzodioxocin structures were identified as lignin interunit linkages, largely independent of harvest times and with β-aryl ethers being expectedly dominant. Carrots contain guaiacyl-rich lignins that incorporate increasing amounts of p-hydroxybenzoate with maturation. All other lignin characteristics appear to be widely independent of harvest times. © 2017 Society of Chemical Industry.

Storage related changes of cell wall based dietary fiber components of broccoli (Brassica oleracea var. italica) stems

Storage related changes in the cell wall composition potentially affect the texture of plant-based foods and the physiological effects of cell wall based dietary fiber components. Therefore, a detailed characterization of cell wall polysaccharides and lignins from broccoli stems was performed. Freshly harvested broccoli and broccoli stored at 20°C and 1°C for different periods of time were analyzed. Effects on dietary fiber contents, polysaccharide composition, and on lignin contents/composition were much more pronounced during storage at 20°C than at 1°C. During storage, insoluble dietary fiber contents of broccoli stems increased up to 13%. Storage related polysaccharide modifications include an increase of the portions of cellulose, xylans, and homogalacturonans and a decrease of the neutral pectic side-chains arabinans and galactans. Broccoli stem lignins are generally rich in guaiacyl units. Lignins from freshly harvested broccoli stems contain slightly larger amounts of p-hydroxyphenyl units than syringyl units. Syringyl units are predominantly incorporated into the lignin polymers during storage, resulting in increased acetyl bromide soluble lignin contents. NMR-based analysis of the interunit linkage types of broccoli stem lignins revealed comparably large portions of resinol structures for a guaiacyl rich lignin. Incorporation of syringyl units into the polymers over storage predominantly occurs through β-O-4-linkages.

Identification of lignin-deficient Brachypodium distachyon (L.) Beauv. mutants induced by gamma radiation.

Brachypodium distachyon (L.) Beauv. is a monocotyledonous model plant that has been studied to understand a range of biological phenomena for lignocellulosic bioethanol feedstocks and other cereal crops. The lignin makes its cell walls recalcitrant to saccharification, constituting the main barrier to lignocellulosic bioethanol production. In this study, lignin-deficient mutants of B. distachyon induced by chronic radiation were selected and the effects of the mutants on fermentable glucose production were identified. Brachypodium distachyon M2 mutants induced by chronically irradiated gamma radiation were screened by the Wiesner test. Lignin-deficient M2 mutants were further confirmed in subsequent M3 and M4 generations by determining acetyl bromide-soluble lignin. The lignin content was significantly reduced in mutant plants 135-2 (by 7.99%), 142-3 (by 13.8%) and 406-1 (by 8.13%) compared with the wild type. Moreover, fermentable glucose was significantly higher in 135-2 (by 23.91%) and 142-3 (by 36.72%) than in the wild type after 72 h of enzymatic hydrolysis. Three lignin-deficient B. distachyon mutants induced by chronically irradiated gamma radiation were obtained. This study will provide fundamental understanding of the B. distachyon cell wall and could contribute to increases in bioethanol production using bioenergy crops. © 2016 Society of Chemical Industry.

Cell Wall Composition and Biomass Recalcitrance Differences Within a Genotypically Diverse Set of Brachypodium distachyon Inbred Lines.

Brachypodium distachyon (Brachypodium) has emerged as a useful model system for studying traits unique to graminaceous species including bioenergy crop grasses owing to its amenability to laboratory experimentation and the availability of extensive genetic and germplasm resources. Considerable natural variation has been uncovered for a variety of traits including flowering time, vernalization responsiveness, and above-ground growth characteristics. However, cell wall composition differences remain underexplored. Therefore, we assessed cell wall-related traits relevant to biomass conversion to biofuels in seven Brachypodium inbred lines that were chosen based on their high level of genotypic diversity as well as available genome sequences and recombinant inbred line (RIL) populations. Senesced stems plus leaf sheaths from these lines exhibited significant differences in acetyl bromide soluble lignin (ABSL), cell wall polysaccharide-derived sugars, hydroxycinnamates content, and syringyl:guaiacyl:p-hydroxyphenyl (S:G:H) lignin ratios. Free glucose, sucrose, and starch content also differed significantly in senesced stems, as did the amounts of sugars released from cell wall polysaccharides (digestibility) upon exposure to a panel of thermochemical pretreatments followed by hydrolytic enzymatic digestion. Correlations were identified between inbred line lignin compositions and plant growth characteristics such as biomass accumulation and heading date (HD), and between amounts of cell wall polysaccharides and biomass digestibility. Finally, stem cell wall p-coumarate and ferulate contents and free-sugars content changed significantly with increased duration of vernalization for some inbred lines. Taken together, these results show that Brachypodium displays substantial phenotypic variation with respect to cell wall composition and biomass digestibility, with some compositional differences correlating with growth characteristics. Moreover, besides influencing HD and biomass accumulation, vernalization was found to affect cell wall composition and free sugars accumulation in some Brachypodium inbred lines, suggesting genetic differences in how vernalization affects carbon flux to polysaccharides. The availability of related RIL populations will allow for the genetic and molecular dissection of this natural variation, the knowledge of which may inform ways to genetically improve bioenergy crop grasses.

Functional co-expression of a fungal ferulic acid esterase and a β-1,4 endoxylanase in Festuca arundinacea (tall fescue) modifies post-harvest cell wall deconstruction.

Improved post-harvest cell wall deconstruction of tall fescue leaves has been demonstrated by in-planta co-expression of a constitutively expressed ferulic acid esterase together with a senescence-induced β-1,4 endoxylanase. Tall fescue plants (Festuca arundinacea) constitutively expressing vacuole- or apoplast-targeted ferulic acid esterase from Aspergillus niger were retransformed with a senescence-induced and apoplast-targeted β-1,4 endo-xylanase from Trichoderma reesei. Enzyme activities in co-expressing plants stabilized after repeated vegetative propagation, with xylanase activity in senescent leaves increasing and ferulic acid esterase activity decreasing after tillering. Plants co-expressing both enzymes in the apoplast, with the lowest levels of ferulate monomers and dimers and the lowest levels of cell wall arabinoxylans, released ten times more cell wall hydroxycinnamic acids and five times more arabinoxylan from the cell wall on autodigestion compared to expression of ferulic acid esterase or xylanase alone. These plants also showed a 31 % increase in cellulase-mediated release of reducing sugars, a 5 % point increase in in vitro dry matter digestibility and a 23 % increase in acetyl bromide-soluble lignin. However, plant growth was adversely affected by expressing FAE in the apoplast, giving plants with narrower shorted leaves, and a 71 % decrease in biomass.

Identification of direct targets of transcription factor MYB46 provides insights into the transcriptional regulation of secondary wall biosynthesis

Secondary wall formation requires coordinated transcriptional regulation of the genes involved in the biosynthesis of the components of secondary wall. Transcription factor (TF) MYB46 (At5g12870) has been shown to function as a central regulator for secondary wall formation in Arabidopsis thaliana, activating biosynthetic genes as well as the TFs involved in the pathways. Recently, we reported that MYB46 directly regulates secondary wall-associated cellulose synthase (CESA4, CESA7, and CESA8) and a mannan synthase (CSLA9) genes. However, it is not known whether MYB46 directly activates the biosynthetic genes for hemicellulose and lignin, which are the other two major components of secondary wall. Based on the observations that the promoter regions of many of the secondary wall biosynthetic genes contain MYB46-binding cis-regulatory motif(s), we hypothesized that MYB46 directly regulates the genes involved in the biosynthesis of the secondary wall components. In this report, we describe several lines of experimental evidence in support of the hypothesis. Electrophoretic mobility shift assay and chromatin immunoprecipitation analysis showed that MYB46 directly binds to the promoters of 13 genes involved in lignin and xylan biosynthesis. We then used steroid receptor-based inducible activation system to confirm that MYB46 directly activates the transcription of the xylan and lignin biosynthetic genes. Furthermore, ectopic up-regulation of MYB46 resulted in a significant increase in xylose and a small increase in lignin content based on acetyl bromide soluble lignin measurements in Arabidopsis. Taken together, we conclude that MYB46 function as a central and direct regulator of the genes involved in the biosynthesis of all three major secondary wall components.

Functional characterization of cinnamyl alcohol dehydrogenase and caffeic acid O-methyltransferase in Brachypodium distachyon.

BackgroundLignin is a significant barrier in the conversion of plant biomass to bioethanol. Cinnamyl alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the pathway of lignin monomer biosynthesis. Brown midrib mutants in Zea mays and Sorghum bicolor with impaired CAD or COMT activity have attracted considerable agronomic interest for their altered lignin composition and improved digestibility. Here, we identified and functionally characterized candidate genes encoding CAD and COMT enzymes in the grass model species Brachypodium distachyon with the aim of improving crops for efficient biofuel production.ResultsWe developed transgenic plants overexpressing artificial microRNA designed to silence BdCAD1 or BdCOMT4. Both transgenes caused altered flowering time and increased stem count and weight. Downregulation of BdCAD1 caused a leaf brown midrib phenotype, the first time this phenotype has been observed in a C3 plant. While acetyl bromide soluble lignin measurements were equivalent in BdCAD1 downregulated and control plants, histochemical staining and thioacidolysis indicated a decrease in lignin syringyl units and reduced syringyl/guaiacyl ratio in the transgenic plants. BdCOMT4 downregulated plants exhibited a reduction in total lignin content and decreased Maule staining of syringyl units in stem. Ethanol yield by microbial fermentation was enhanced in amiR-cad1-8 plants.ConclusionThese results have elucidated two key genes in the lignin biosynthetic pathway in B. distachyon that, when perturbed, may result in greater stem biomass yield and bioconversion efficiency.

CCoAOMT suppression modifies lignin composition in Pinus radiata

A cDNA clone encoding the lignin-related enzyme caffeoyl CoA 3-O-methyltransferase (CCoAOMT) was isolated from a Pinus radiata cDNA library derived from differentiating xylem. Suppression of PrCCoAOMT expression in P.radiata tracheary element cultures affected lignin content and composition, resulting in a lignin polymer containing p-hydroxyphenyl (H), catechyl (C) and guaiacyl (G) units. Acetyl bromide-soluble lignin assays revealed reductions in lignin content of up to 20% in PrCCoAOMT-deficient transgenic lines. Pyrolysis-GC/MS and 2D-NMR studies demonstrated that these reductions were due to depletion of G-type lignin. Correspondingly, the proportion of H-type lignin in PrCCoAOMT-deficient transgenic lines increased, resulting in up to a 10-fold increase in the H/G ratio relative to untransformed controls. 2D-NMR spectra revealed that PrCCoAOMT suppression resulted in formation of benzodioxanes in the lignin polymer. This suggested that phenylpropanoids with an ortho-diphenyl structure such as caffeyl alcohol are involved in lignin polymerization. To test this hypothesis, synthetic lignins containing methyl caffeate or caffeyl alcohol were generated and analyzed by 2D-NMR. Comparison of the 2D-NMR spectra from PrCCoAOMT-RNAi lines and synthetic lignins identified caffeyl alcohol as the new lignin constituent in PrCCoAOMT-deficient lines. The incorporation of caffeyl alcohol into lignin created a polymer containing catechyl units, a lignin type that has not been previously identified in recombinant lignin studies. This finding is consistent with the theory that lignin polymerization is based on a radical coupling process that is determined solely by chemical processes.

Cell wall-bound peroxidase activity and lignin formation in azuki bean epicotyls grown under hypergravity conditions

The effects of accelerated gravity stimuli on the cell wall-bound peroxidase activity and the lignin content were investigated along epicotyls of azuki bean (Vigna angularis) seedlings. The endogenous growth occurred primarily in the upper regions of the epicotyl, but no growth was detected in the middle or basal regions. Hypergravity treatment at 300g for 6h suppressed elongation growth and stimulated lateral expansion of the upper regions. The content of acetyl bromide-soluble lignin increased gradually from the apical to the basal regions of epicotyls. Hypergravity treatment stimulated the increase in the lignin content in epicotyls, particularly in the middle and basal regions. The peroxidase activity in the protein fraction extracted with a high ionic strength buffer from the cell wall preparation also increased gradually toward the basal region, and hypergravity treatment increased the activity in all epicotyl regions. There was a close correlation between the lignin content and the enzyme activity. These results suggest that hypergravity increases the activity of cell wall-bound peroxidase followed by increases of the lignin formation in epicotyl cell walls, which may contribute to increasing the rigidity of cell walls against the gravitational force.

Suppression of 4-coumarate-CoA ligase in the coniferous gymnosperm Pinus radiata.

Severe suppression of 4-coumarate-coenzyme A ligase (4CL) in the coniferous gymnosperm Pinus radiata substantially affected plant phenotype and resulted in dwarfed plants with a "bonsai tree-like" appearance. Microscopic analyses of stem sections from 2-year-old plants revealed substantial morphological changes in both wood and bark tissues. This included the formation of weakly lignified tracheids that displayed signs of collapse and the development of circumferential bands of axial parenchyma. Acetyl bromide-soluble lignin assays and proton nuclear magnetic resonance studies revealed lignin reductions of 36% to 50% in the most severely affected transgenic plants. Two-dimensional nuclear magnetic resonance and pyrolysis-gas chromatography-mass spectrometry studies indicated that lignin reductions were mainly due to depletion of guaiacyl but not p-hydroxyphenyl lignin. 4CL silencing also caused modifications in the lignin interunit linkage distribution, including elevated beta-aryl ether (beta-O-4 unit) and spirodienone (beta-1) levels, accompanied by lower phenylcoumaran (beta-5), resinol (beta-beta), and dibenzodioxocin (5-5/beta-O-4) levels. A sharp depletion in the level of saturated (dihydroconiferyl alcohol) end groups was also observed. Severe suppression of 4CL also affected carbohydrate metabolism. Most obvious was an up to approximately 2-fold increase in galactose content in wood from transgenic plants due to increased compression wood formation. The molecular, anatomical, and analytical data verified that the isolated 4CL clone is associated with lignin biosynthesis and illustrated that 4CL silencing leads to complex, often surprising, physiological and morphological changes in P. radiata.

Comparison of the acetyl bromide spectrophotometric method with other analytical lignin methods for determining lignin concentration in forage samples.

Present analytical methods to quantify lignin in herbaceous plants are not totally satisfactory. A spectrophotometric method, acetyl bromide soluble lignin (ABSL), has been employed to determine lignin concentration in a range of plant materials. In this work, lignin extracted with acidic dioxane was used to develop standard curves and to calculate the derived linear regression equation (slope equals absorptivity value or extinction coefficient) for determining the lignin concentration of respective cell wall samples. This procedure yielded lignin values that were different from those obtained with Klason lignin, acid detergent acid insoluble lignin, or permanganate lignin procedures. Correlations with in vitro dry matter or cell wall digestibility of samples were highest with data from the spectrophotometric technique. The ABSL method employing as standard lignin extracted with acidic dioxane has the potential to be employed as an analytical method to determine lignin concentration in a range of forage materials. It may be useful in developing a quick and easy method to predict in vitro digestibility on the basis of the total lignin content of a sample.

Correlação entre digestibilidade in vitro da parede celular e três métodos analíticos para a avaliação quantitativa da lignina

Três métodos analíticos para a estimativa do conteúdo de lignina na parede celular, em diferentes frações vegetais, de sete espécies forrageiras, foram comparados. Os métodos empregados foram: lignina em detergente ácido (LDA), lignina permanganato de potássio e lignina solúvel em brometo de acetila (LSBA). Para o método espectrofotométrico (LSBA), foi utilizado como padrão de referência a lignina extraída da planta através do próprio reagente brometo de acetila. Não houve concordância entre os três métodos, sendo que, no geral, o método da LSBA mostrou valores mais elevados que os outros dois métodos e o método da LDA, menores valores. Na maioria das amostras, a concentração de lignina foi superior para a parede celular oriunda de planta madura em relação àquela proveniente de planta jovem, assim como o teor de lignina foi maior para a fração colmo em relação à fração folha. As leguminosas tenderam a apresentar teores mais elevados de lignina pelos métodos gravimétricos que as gramíneas, independentemente do estádio de maturidade. Entretanto, essa tendência não foi observada para o método LSBA, particularmente no estádio maduro. Preparações de parede celular foram fermentadas in vitro e os dados correlacionados com os teores de lignina obtidos por intermédio dos três métodos. O método LSBA exibiu os menores coeficientes de correlação, para a maioria das amostras, sugerindo que este método deve ser melhor avaliado.

Extração da lignina e emprego da mesma em curvas de calibração para a mensuração da lignina em produtos vegetais

O objetivo deste experimento foi a extração da lignina e seu uso nas curvas de calibração para determinar a concentração de lignina em produtos vegetais. Dentre os métodos analíticos utilizados para a mensuração da lignina, pode-se mencionar o método "lignina solúvel em brometo de acetila - LSBA", no qual a lignina é solubilizada em uma solução de brometo de acetila a 25% em ácido acético glacial e, depois, lida no comprimento de onda a 280 nm. Entretanto, todo método espectrofotométrico requer o emprego de um padrão de referência confiável; neste experimento utilizou-se como padrão de referência a lignina da planta forrageira extraída com o emprego do próprio brometo de acetila. Quantificou-se a lignina presente em quatro amostras de forrageiras, em dois estádios de maturidade e, ainda, em duas amostras de madeiras, comparando-se os dados com outros dois métodos de determinação da lignina (lignina em detergente ácido - LDA e lignina permanganato de potássio - LPer). Os três métodos não foram concordantes entre si, sendo que, para praticamente todas as amostras, o método da LSBA mostrou valores mais elevados que os outros dois métodos. Para cada amostra analisada, foram confeccionados uma curva-padrão e um espectrograma na faixa de comprimento de luz de 240 a 320 nm. A análise dessas curvas e dos espectrogramas indicou diferenças qualitativas entre as amostras, em função não apenas da espécie botânica, mas também do estádio de maturidade da planta.