Infrared Nanospectroscopy Reveals the Chemical Nature of Pit Membranes in Water-Conducting Cells of the Plant Xylem.

In the xylem of angiosperm plants, microscopic pits through the secondary cell walls connect the water-conducting vessels. Cellulosic meshes originated from primary walls, and middle lamella between adjacent vessels, called the pit membrane, separates one conduit from another. The intricate structure of the nano-sized pores in pit membranes enables the passage of water under negative pressure without hydraulic failure due to obstruction by gas bubbles (i.e. embolism) under normal conditions or mild drought stress. Since the chemical composition of pit membranes affects embolism formation and bubble behavior, we directly measured pit membrane composition in Populus nigra wood. Here, we characterized the chemical composition of cell wall structures by synchrotron infrared nanospectroscopy and atomic force microscopy-infrared nanospectroscopy with high spatial resolution. Characteristic peaks of cellulose, phenolic compounds, and proteins were found in the intervessel pit membranes of P. nigra wood. In addition, the vessel to parenchyma pit membranes and developing cell walls of the vascular cambium showed clear signals of cellulose, proteins, and pectin. We did not find a distinct peak of lignin and other compounds in these structures. Our investigation of the complex chemical composition of intervessel pit membranes furthers our understanding of the flow of water and bubbles between neighboring conduits. The advances presented here pave the way for further label-free studies related to the nanochemistry of plant cell components.

Optimization of RT-PCR reactions in studies with genes of lignin biosynthetic route in Saccharum spontaneum.

Saccharum spontaneum has been used for the development of energy cane a crop aimed to be used for the production of second-generation ethanol, or lignocellulosic ethanol. Lignin is a main challenge in the conversion of cell wall sugars into ethanol. In our studies to isolate the genes the lignin biosynthesis in S. spontaneum we have had great difficulty in RT-PCR reactions. Thus, we evaluated the effectiveness of different additives in the amplification of these genes. While COMT and CCoAOMT genes did not need any additives for other genes there was no amplification (HCT, F5H, 4CL and CCR) or the yield was very low (CAD and C4H). The application of supplementary cDNA was enough to overcome the non-specificity and low yield for C4H and C3H, while the addition of 0.04% BSA + 2% formamide was effective to amplify 4CL, CCR, F5H and CCR. HCT was amplified only by addition of 0.04% BSA + 2% formamide + 0.1 M trehalose and amplification of PAL was possible with addition of 2% of DMSO. Besides optimization of expression assays, the results show that additives can act independently or synergistically.

Luxurious Nitrogen Fertilization of Two Sugar Cane Genotypes Contrasting for Lignin Composition Causes Changes in the Stem Proteome Related to Carbon, Nitrogen, and Oxidant Metabolism but Does Not Alter Lignin Content.

Sugar cane is an important crop for sugar and biofuel production. Its lignocellulosic biomass represents a promising option as feedstock for second-generation ethanol production. Nitrogen fertilization can affect differently tissues and its biopolymers, including the cell-wall polysaccharides and lignin. Lignin content and composition are the most important factors associated with biomass recalcitrance to convert cell-wall polysaccharides into fermentable sugars. Thus it is important to understand the metabolic relationship between nitrogen fertilization and lignin in this feedstock. In this study, a large-scale proteomics approach based on GeLC-MS/MS was employed to identify and relatively quantify proteins differently accumulated in two contrasting genotypes for lignin composition after excessive nitrogen fertilization. From the ∼1000 nonredundant proteins identified, 28 and 177 were differentially accumulated in response to nitrogen from IACSP04-065 and IACSP04-627 lines, respectively. These proteins were associated with several functional categories, including carbon metabolism, amino acid metabolism, protein turnover, and oxidative stress. Although nitrogen fertilization has not changed lignin content, phenolic acids and lignin composition were changed in both species but not in the same way. Sucrose and reducing sugars increased in plants of the genotype IACSP04-065 receiving nitrogen.

A simple protocol to determine lignin S/G ratio in plants by UHPLC-MS.

A simple extraction protocol and an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) method for the determination of the syringyl/guaiacyl (S/G) ratio in lignin is reported herein. The method was entirely developed using stems of three Eucalyptus species, which were hydrolyzed with NaOH and partitioned with ethyl ether; vanillin (from the G monomer) and syringaldehyde (from S monomer) were quantified. The S/G ratios obtained were comparable to those usually reported for eucalyptus. The data for one of the eucalyptus species were compared with those obtained with a widely accepted method using thioacidolysis and gas chromatography-mass spectrometry (GC-MS). The method was also applied to sugarcane and showed to be reliable. The yield of the NaOH hydrolysis of the monolignols ranged from 89.94 to 95.69%, with more than 77.12% of recuperation in the liquid-liquid extraction. The whole analytical procedure was validated, achieving results with less than 4.38% of variation. The lowest LOD and LOQ were 0.01 and 0.05 µg/mL, respectively. In addition, the method combines reliability and a fast and direct quantification.

Mass spectrometry imaging: an expeditious and powerful technique for fast in situ lignin assessment in Eucalyptus.

Plant biomass has been suggested as an alternative to produce bioethanol. The recalcitrance of plant biomass to convert cellulose into simpler carbohydrates used in the fermentation process is partially due to lignin, but the standard methods used to analyze lignin composition frequently use toxic solvents and are laborious and time-consuming. MS imaging was used to study lignin in Eucalyptus, since this genus is the main source of cellulose in the world. Hand-cut sections of stems of two Eucalyptus species were covered with silica and directly analyzed by matrix-assisted laser sesorption ionization (MALDI)-imaging mass spectrometry (MS). Information available in the literature about soluble lignin subunits and structures were used to trace their distribution in the sections and using a software image a relative quantification could be made. Matrixes routinely used in MALDI-imaging analysis are not satisfactory to analyze plant material and were efficiently substituted by thin layer chromatography (TLC) grade silica. A total of 22 compounds were detected and relatively quantified. It was also possible to establish a proportion between syringyl and guaiacyl monolignols, characteristic for each species. Because of the simple way that samples are prepared, the MALDI-imaging approach presented here can replace, in routine analysis, complex and laborious MS methods in the study of lignin composition.

Lignification in sugarcane: biochemical characterization, gene discovery, and expression analysis in two genotypes contrasting for lignin content.

Sugarcane (Saccharum spp.) is currently one of the most efficient crops in the production of first-generation biofuels. However, the bagasse represents an additional abundant lignocellulosic resource that has the potential to increase the ethanol production per plant. To achieve a more efficient conversion of bagasse into ethanol, a better understanding of the main factors affecting biomass recalcitrance is needed. Because several studies have shown a negative effect of lignin on saccharification yield, the characterization of lignin biosynthesis, structure, and deposition in sugarcane is an important goal. Here, we present, to our knowledge, the first systematic study of lignin deposition during sugarcane stem development, using histological, biochemical, and transcriptional data derived from two sugarcane genotypes with contrasting lignin contents. Lignin amount and composition were determined in rind (outer) and pith (inner) tissues throughout stem development. In addition, the phenolic metabolome was analyzed by ultra-high-performance liquid chromatography-mass spectrometry, which allowed the identification of 35 compounds related to the phenylpropanoid pathway and monolignol biosynthesis. Furthermore, the Sugarcane EST Database was extensively surveyed to identify lignin biosynthetic gene homologs, and the expression of all identified genes during stem development was determined by quantitative reverse transcription-polymerase chain reaction. Our data provide, to our knowledge, the first in-depth characterization of lignin biosynthesis in sugarcane and form the baseline for the rational metabolic engineering of sugarcane feedstock for bioenergy purposes.

A model system to study the lignification process in Eucalyptus globulus.

Recalcitrance of plant biomass is closely related to the presence of the phenolic heteropolymer lignin in secondary cell walls, which has a negative effect on forage digestibility, biomass-to-biofuels conversion and chemical pulping. The genus Eucalyptus is the main source of wood for pulp and paper industry. However, when compared to model plants such as Arabidopsis thaliana and poplar, relatively little is known about lignin biosynthesis in Eucalyptus and only a few genes were functionally characterized. An efficient, fast and inexpensive in vitro system was developed to study lignification in Eucalyptus globulus and to evaluate the potential role of candidate genes in this biological process. Seedlings were grown in four different conditions, in the presence or absence of light and with or without sucrose in the growth medium, and several aspects of lignin metabolism were evaluated. Our results showed that light and, to a lesser extent, sucrose induced lignin biosynthesis, which was followed by changes in S/G ratio, lignin oligomers accumulation and gene expression. In addition, higher total peroxidase activity and differential isoperoxidase profile were observed when seedlings were grown in the presence of light and sucrose. Peptide sequencing allowed the identification of differentially expressed peroxidases, which can be considered potential candidate class III peroxidases involved in lignin polymerization in E. globulus.

ShF5H1 overexpression increases syringyl lignin and improves saccharification in sugarcane leaves.

The agricultural sugarcane residues, bagasse and straws, can be used for second-generation ethanol (2GE) production by the cellulose conversion into glucose (saccharification). However, the lignin content negatively impacts the saccharification process. This polymer is mainly composed of guaiacyl (G), hydroxyphenyl (H), and syringyl (S) units, the latter formed in the ferulate 5-hydroxylase (F5H) branch of the lignin biosynthesis pathway. We have generated transgenic lines overexpressing ShF5H1 under the control of the C4H (cinnamate 4-hydroxylase) rice promoter, which led to a significant increase of up to 160% in the S/G ratio and 63% in the saccharification efficiency in leaves. Nevertheless, the content of lignin was unchanged in this organ. In culms, neither the S/G ratio nor sucrose accumulation was altered, suggesting that ShF5H1 overexpression would not affect first-generation ethanol production. Interestingly, the bagasse showed a significantly higher fiber content. Our results indicate that the tissue-specific manipulation of the biosynthetic branch leading to S unit formation is industrially advantageous and has established a foundation for further studies aiming at refining lignin modifications. Thus, the ShF5H1 overexpression in sugarcane emerges as an efficient strategy to improve 2GE production from straw.

Expression of SofLAC, a new laccase in sugarcane, restores lignin content but not S:G ratio of Arabidopsis lac17 mutant.

Lignin is a complex phenolic heteropolymer deposited in the secondarily thickened walls of specialized plant cells to provide strength for plants to stand upright and hydrophobicity to conducting cells for long-distance water transport. Although essential for plant growth and development, lignin is the major plant cell-wall component responsible for biomass recalcitrance to industrial processing. Peroxidases and laccases are generally thought to be responsible for lignin polymerization, but, given their broad substrate specificities and large gene families, specific isoforms involved in lignification are difficult to identify. This study used a combination of co-expression analysis, tissue/cell-type-specific expression analysis, and genetic complementation to correlate a sugarcane laccase gene, SofLAC, to the lignification process. A co-expression network constructed from 37 cDNA libraries showed that SofLAC was coordinately expressed with several phenylpropanoid biosynthesis genes. Tissue-specific expression analysis by quantitative RT-PCR showed that SofLAC was expressed preferentially in young internodes and that expression levels decrease with stem maturity. Cell-type-specific expression analysis by in situ hybridization demonstrated the localization of SofLAC mRNA in lignifying cell types, mainly in inner and outer portions of sclerenchymatic bundle sheaths. To investigate whether SofLAC is able to oxidize monolignols during lignification, the Arabidopsis lac17 mutant, which has reduced lignin levels, was complemented by expressing SofLAC under the control of the Arabidopsis AtLAC17 promoter. The expression of SofLAC restored the lignin content but not the lignin composition in complemented lac17 mutant lines. Taken together, these results suggest that SofLAC participates in lignification in sugarcane.

Analysis of soluble lignin in sugarcane by ultrahigh performance liquid chromatography-tandem mass spectrometry with a do-it-yourself oligomer database.

Lignin is a polymer found in the cell wall of plants and is one of the main obstacles to the implementation of second-generation ethanol production because it confers the recalcitrance of the lignocellulosic material. The recalcitrance of biomass is affected by the amount of lignin, by its monomer composition, and the way the monomers are arranged in the plant cell wall. Analysis of lignin structure demands mass spectrometry analysis, and identification of oligomers is usually based on libraries produced by laborious protocols. A robust method to build a do-it-yourself lignin oligomer library was tested. This library can be built using commercially available enzymes, standards, and reagents and is relatively easy to accomplish. An ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the separation and characterization of monomers and oligomers was developed and was equally applicable to the synthetic lignin and to soluble lignin extracted from a sample of sugar cane.

Visualization of Suberization and Lignification in Sugarcane.

Cell wall biopolymers are major factors responsible for the high recalcitrance of sugarcane biomass. The study of suberization and lignification mechanisms in sugarcane and of the networks that control biosynthesis of these polymers will contribute to the biotechnological improvement of this crop. Here, we describe experiments that allow the visualization of the suberization and lignification mechanism in response to mechanical injury in sugarcane.

Isolation of Promoters and Transcription Factors Involved in the Regulation of Lignin Biosynthesis in Saccharum Species.

Sugarcane bagasse has received attention as a raw material for the production of second-generation ethanol (E2G). However, its use is limited because of the cell wall recalcitrance, mostly conferred by lignin. Recently our knowledge of the genes coding for the enzymes of the lignin biosynthesis pathway has increased; however, still little is known about the transcription factors controlling the expression of these genes in sugarcane. Here we describe protocols to optimize the isolation of the promoters of the lignin biosynthetic genes ShCAD8, ShCOMT and ShF5H and the transcription factors (TFs) ShMYB85 and ShMYB58/63 in Saccharum species. To confirm whether these TFs are able to activate the target promoters, a transactivation assay in BY2 protoplasts of Nicotiana tabacum is also detailed.

Abiotic and biotic stresses and changes in the lignin content and composition in plants.

Lignin is a polymer of phenylpropanoid compounds formed through a complex biosynthesis route, represented by a metabolic grid for which most of the genes involved have been sequenced in several plants, mainly in the model-plants Arabidopsis thaliana and Populus. Plants are exposed to different stresses, which may change lignin content and composition. In many cases, particularly for plant-microbe interactions, this has been suggested as defence responses of plants to the stress. Thus, understanding how a stressor modulates expression of the genes related with lignin biosynthesis may allow us to develop study-models to increase our knowledge on the metabolic control of lignin deposition in the cell wall. This review focuses on recent literature reporting on the main types of abiotic and biotic stresses that alter the biosynthesis of lignin in plants.

Correlations between lignin content and structural robustness in plants revealed by X-ray ptychography.

Lignin is a heterogeneous aromatic polymer responsible for cell wall stiffness and protection from pathogen attack. However, lignin represents a bottleneck to biomass degradation due to its recalcitrance related to the natural cell wall resistance to release sugars for fermentation or further processing. A biological approach involving genetics and molecular biology was used to disrupt lignin pathway synthesis and decrease lignin deposition. Here, we imaged three-dimensional fragments of the petioles of wild type and C4H lignin mutant Arabidopsis thaliana plants by synchrotron cryo-ptychography. The three-dimensional images revealed the heterogeneity of vessels, parenchyma, and fibre cell wall morphologies, highlighting the relation between disturbed lignin deposition and vessel implosion (cell collapsing and obstruction of water flow). We introduce a new parameter to accurately define cell implosion conditions in plants, and we demonstrate how cryo-ptychographic X-ray computed tomography (cryo-PXCT) provides new insights for plant imaging in three dimensions to understand physiological processes.

Should I stay or should I go: are chlorogenic acids mobilized towards lignin biosynthesis?

Chlorogenic acids (CGAs) and the biopolymer lignin are both products of the phenylpropanoid pathway. Whereas CGAs have been reported to play a role during stress responses, lignin is a major component of secondary cell walls, providing physical strength and hydrophobicity to supportive and water-conducting tissues. Because the chemical structure of CGAs largely resembles those of some lignin intermediates and because CGAs can be converted back to hydroxycinnamoyl-CoAs in vitro, CGAs have been considered authentic intermediates of the lignin biosynthetic pathway. However, it is still unclear whether and how the CGA pool can be channeled towards the production of lignin monomers in response to developmental or environmental signals. Comprehensive studies on the catalytic activity of recombinant enzymes together with functional characterizations in planta have been very useful in understanding the potential interdependence between these two metabolic routes. Here we present the current understanding on CGA metabolism and discuss the biochemical and molecular evidence of the metabolic re-routing of CGAs towards lignin.

Deposition of lignin in four species of Saccharum.

We used primers designed on conserved gene regions of several species to isolate the most expressed genes of the lignin pathway in four Saccharum species. S. officinarum and S. barberi have more sucrose in the culms than S. spontaneum and S. robustum, but less polysaccharides and lignin in the cell wall. S. spontaneum, and S. robustum had the lowest S/G ratio and a lower rate of saccharification in mature internodes. Surprisingly, except for CAD, 4CL, and CCoAOMT for which we found three, two, and two genes, respectively, only one gene was found for the other enzymes and their sequences were highly similar among the species. S. spontaneum had the highest expression for most genes. CCR and CCoAOMT B presented the highest expression; 4CL and F5H showed increased expression in mature tissues; C3H and CCR had higher expression in S. spontaneum, and one of the CADs isolated (CAD B) had higher expression in S. officinarum. The similarity among the most expressed genes isolated from these species was unexpected and indicated that lignin biosynthesis is conserved in Saccharum including commercial varieties Thus the lignin biosynthesis control in sugarcane may be only fully understood with the knowledge of the promotor region of each gene.

Insights into temperature modulation of the Eucalyptus globulus and Eucalyptus grandis antioxidant and lignification subproteomes.

Eucalyptus grandis and Eucalyptus globulus are among the most widely cultivated trees, differing in lignin composition and plantation areas, as E. grandis is mostly cultivated in tropical regions while E. globulus is preferred in temperate areas. As temperature is a key modulator in plant metabolism, a large-scale proteome analysis was carried out to investigate changes in the antioxidant system and the lignification metabolism in plantlets grown at different temperatures. Our strategy allowed the identification of 3111 stem proteins. A total of 103 antioxidant proteins were detected in the stems of both species. Hierarchical clustering revealed that alterations in the antioxidant proteins are more prominent when Eucalyptus seedlings were exposed to high temperature and that the superoxide isoforms coded by the gene Eucgr.B03930 are the most abundant antioxidant enzymes induced by thermal stimulus. Regarding the lignin biosynthesis, our proteomics approach resulted in the identification of 13 of the 17 core proteins involved in this metabolism, corroborating with gene predictions and the proposed lignin toolbox. Quantitative analyses revealed significant differences in 8 protein isoforms, including the ferulate 5-hydroxylase isoform F5H1, a key enzyme in catalyzing the synthesis of sinapyl alcohol, and the cinnamyl alcohol dehydrogenase isoform CAD2, the last enzyme in monolignol biosynthesis. Data are available via ProteomeXchange with identifier PXD005743.

Water stress alters lignin content and related gene expression in two sugarcane genotypes.

The lignin deposition in the stem of two sugarcane genotypes was assessed on exposure to water stress. The lignin content and the morphoanatomical characterization of the stem indicated that IACSP94-2094 plants are more lignified than those of IACSP95-5000 genotype, under normal water supply conditions, which was especially associated with higher lignin contents in the rind of mature internodes. Water deficit had negative impact on the biomass production, mostly with IACSP94-2094 plants, possibly due to stress severity or higher susceptibility of that genotype during the stem-lengthening phase. Water deficit led to significant alterations in the expression levels of lignin biosynthesis genes and led to an approximate 60% increase of lignin content in the rind of young internodes in both genotypes. It is concluded that the young rind region was more directly affected by water stress and, depending on the genotype, a higher lignin accumulation may occur in the stem, thus implying lower quality biomass for bioethanol production.

Large-Scale Transcriptome Analysis of Two Sugarcane Genotypes Contrasting for Lignin Content.

Sugarcane is an important crop worldwide for sugar and first generation ethanol production. Recently, the residue of sugarcane mills, named bagasse, has been considered a promising lignocellulosic biomass to produce the second-generation ethanol. Lignin is a major factor limiting the use of bagasse and other plant lignocellulosic materials to produce second-generation ethanol. Lignin biosynthesis pathway is a complex network and changes in the expression of genes of this pathway have in general led to diverse and undesirable impacts on plant structure and physiology. Despite its economic importance, sugarcane genome was still not sequenced. In this study a high-throughput transcriptome evaluation of two sugarcane genotypes contrasting for lignin content was carried out. We generated a set of 85,151 transcripts of sugarcane using RNA-seq and de novo assembling. More than 2,000 transcripts showed differential expression between the genotypes, including several genes involved in the lignin biosynthetic pathway. This information can give valuable knowledge on the lignin biosynthesis and its interactions with other metabolic pathways in the complex sugarcane genome.

Optimization of RT-PCR reactions in studies with genes of lignin biosynthetic route in Saccharum spontaneum

ABSTRACT Saccharum spontaneum has been used for the development of energy cane a crop aimed to be used for the production of second-generation ethanol, or lignocellulosic ethanol. Lignin is a main challenge in the conversion of cell wall sugars into ethanol. In our studies to isolate the genes the lignin biosynthesis in S. spontaneum we have had great difficulty in RT-PCR reactions. Thus, we evaluated the effectiveness of different additives in the amplification of these genes. While COMT and CCoAOMT genes did not need any additives for other genes there was no amplification (HCT, F5H, 4CL and CCR) or the yield was very low (CAD and C4H). The application of supplementary cDNA was enough to overcome the non-specificity and low yield for C4H and C3H, while the addition of 0.04% BSA + 2% formamide was effective to amplify 4CL, CCR, F5H and CCR. HCT was amplified only by addition of 0.04% BSA + 2% formamide + 0.1 M trehalose and amplification of PAL was possible with addition of 2% of DMSO. Besides optimization of expression assays, the results show that additives can act independently or synergistically.