Possible approaches for studying three dimensional structure of lignin

Now we are attempting to apply non destructive analysis from evaluation tests or failure analysis to acceptance tests or production tests. Needless to say non destructive analysis has an advantage of conserving the state of samples and the reducing the time of analysis as compared to conventional methods with destructive physical analysis. Moreover, we are paying attention to the following reasons for nondestructive physical analysis. It is difficult to keep the reproducibility of the analysis because of the high skill level required for destructive physical analysis. On the other hand, high reproducibility can be easily achieved by fixing the condition or parameters of the device during nondestructive analysis when performed by tools like X-ray. Moreover, we expect that neither the analytical result nor the quality of the nondestructive analysis depends upon the worker's capability. In this paper we will discuss the following two items from the viewpoint of quality assurance. 1. The method of the screening for fake parts (1) The procedure flow for the production discontinued parts (2) The comparison and examination between the diagnostic using X-ray computed tomography (X-ray CT) images and various examinations (3) Other observation cases using X-ray CT images 2. Effectiveness and consideration in reliability evaluation test using X-ray CT image (1) Comparison of observation cases with a variety of jointing points in parts (2) Consideration of application of nondestructive observation technique in reliability test Use of X-ray CT images is effective in diagnosing the quality of the product or the process. Moreover, we find that use of X-ray CT images is effective for the improvement of the reproducibility of the evaluation examination. Then we find that use of X-ray CT images can reduce the time of evaluation examination too.

Panax notoginseng PnMYB2 is a transcriptional activator of primary and secondary cell wall formation by promoting the PCW-specific gene CesA3 and key lignin biosynthetic gene CCoAOMT1, respectively. R2R3-MYB transcription factors play important roles in regulation secondary cell wall (SCW) formation. However, there are few reports on the functions of MYB transcription factors which involved in both primary cell wall (PCW) and SCW formation. Here, we isolated an R2R3-MYB transcription factor, PnMYB2, from Panax notoginseng roots which are widely used in Chinese traditional medicines and contain abundant cellulose and lignin. The expression pattern of PnMYB2 was similar to the accumulation pattern of cellulose and lignin contents in different organs. PnMYB2 localized in the nucleus and may function as a transcriptional activator. Overexpression of PnMYB2 in Arabidopsis thaliana enhanced cellulose and lignin biosynthesis, and remarkably increased thickness of PCW and SCW in the stem of transgenic plants compared with wild-type plants. The expression levels of genes associated with PCW-specific cellulose synthase (CesA) genes and key SCW-specific lignin biosynthetic genes were significantly increased in PnMYB2-overexpressing plants compared to the wild type plants. Furthermore, yeast one-hybrid, dual-luciferase reporter assays and electrophoretic mobility shift assays (EMSA) results verified that PnMYB2 could bind and activate the promoters of AtCesA3 and PnCesA3, which are the PCW-specific cellulose biosynthetic genes, and AtCCoAOMT1 and PnCCoAOMT1, which are the key lignin biosynthetic genes. These results demonstrated the central role of PnMYB2 in PCW-specific cellulose formation and SCW-specific lignin biosynthesis.

The feruloylarabinoxylan deposition was initiated at the formation of the secondary cell wall, especially S2 layer in moso bamboo, which may affect crosslinking between cell wall components and plant growth. Hemicelluloses, major components of plant cell walls that are hydrogen bonded to cellulose and covalently bound to lignin, are crucial determinants of cell wall properties. Especially in commelinid monocotyledons, arabinoxylan is often esterified with ferulic acid, which is essential to crosslinking with cell wall components. However, the deposition patterns and localization of ferulic acid during cell wall formation remain unclear. In this study, developing moso bamboo (Phyllostachys pubescens) culms were used to elucidate deposition patterns of hemicelluloses including feruloylarabinoxylan. Ferulic acid content peaked with cessation of elongation growth, and thereafter decreased and remained stable as culm development proceeded. During primary cell wall (PCW) formation, xyloglucan and (1,3;1,4)-β-glucan signals were detected in all tissues. Along with culm development, arabinoxylan and feruloylarabinoxylan signals were sequentially observed in the protoxylem, vascular fibers and metaxylem, and parenchyma. Feruloylarabinoxylan signals were observed slightly later than arabinoxylan signals. Arabinoxylan signals were observed throughout the compound middle lamella and secondary cell wall (SCW), whereas the feruloylarabinoxylan signal was localized to the S2 layer of the SCW. These results indicate that the biosynthesis of hemicelluloses is regulated in accordance with cell wall layers. Feruloylarabinoxylan deposition may be initiated at the formation of SCW, especially S2 layer formation. Ferulic acid-mediated linkages of arabinoxylan-arabinoxylan and arabinoxylan-lignin would arise during SCW formation with the cessation of elongation growth.

Lettuce are vegetables with a high commercial value and a short cycle production, which requires precise managements to guarantee a profitable production. For this, the nutritional providing is an important factor, highlining the nitrogen, a macronutrient considered essential for the proper development of plants due to its participation in the composition of the main plant molecules, such as chlorophyll. In this sense, non-destructive strategies to monitor the balance nutrition is fundamental to avoid deficiency or excess of nutrients during the lettuce cycling, and the agriculture 4.0, brings to light new technological means to achieve this goal. This work aimed to verify the potential to use a portable spectrophotometer to estimate N shoot in lettuce, as a non-destructive, precise, quickly, cheap, waste free option to be operated by the lettuce farmers, on farm, for the N monitoring, based on the destructive laboratory analysis for N shoot and chlorophyll, and the widely used sensor chlorophyll meter (SPAD). For this, a greenhouse experiment was conducted with lettuce cropped under N levels input, corresponding to 0, 25, 50, 75, 100 and 125% of the lettuce recommendation. The estimation for N in the lettuce shoot was performed by destructive analysis: i) shoot N; ii) chlorophyll and non-destructive analysis: iii) chlorophyll meter SPAD and iv) a portable spectrophotometer. Non-destructive measurements were performed 3 times at: 20, 23 and 26 days after transplanting (DAT). The lettuce harvest occurred 28 DAT, to determine N shoot uptake and chlorophyll, using a destructive way, and lettuce production. The estimation of N shoot in lettuce using the portable spectrophotometer showed a high correlation to the standard destructive laboratory analysis and the chlorophyll meter (SPAD), showing high explanation of the data and so a high potential to estimate N shoot in lettuce using the proposed portable spectrophotometer as an optimum non-destructive, precise, quickly, cheap, waste free option to be operated by the lettuce farmers, on farm, for the N monitoring.

BackgroundLeucine-rich repeat extensins (LRXs) are extracellular proteins consisting of an N-terminal leucine-rich repeat (LRR) domain and a C-terminal extensin domain containing the typical features of this class of structural hydroxyproline-rich glycoproteins (HRGPs). The LRR domain is likely to bind an interaction partner, whereas the extensin domain has an anchoring function to insolubilize the protein in the cell wall. Based on the analysis of the root hair-expressed LRX1 and LRX2 of Arabidopsis thaliana, LRX proteins are important for cell wall development. The importance of LRX proteins in non-root hair cells and on the structural changes induced by mutations in LRX genes remains elusive.ResultsThe LRX gene family of Arabidopsis consists of eleven members, of which LRX3, LRX4, and LRX5 are expressed in aerial organs, such as leaves and stem. The importance of these LRX genes for plant development and particularly cell wall formation was investigated. Synergistic effects of mutations with gradually more severe growth retardation phenotypes in double and triple mutants suggest a similar function of the three genes. Analysis of cell wall composition revealed a number of changes to cell wall polysaccharides in the mutants.ConclusionsLRX3, LRX4, and LRX5, and most likely LRX proteins in general, are important for cell wall development. Due to the complexity of changes in cell wall structures in the lrx mutants, the exact function of LRX proteins remains to be determined. The increasingly strong growth-defect phenotypes in double and triple mutants suggests that the LRX proteins have similar functions and that they are important for proper plant development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0548-8) contains supplementary material, which is available to authorized users.

Synchronizing cell growth, division and DNA replication is an essential property of all living cells. Accurate coordination of these cellular events is especially crucial for bacteria, which can grow rapidly and undergo multifork replication. Here we show that the metabolic protein ManA, which is a component of mannose phosphotransferase system, participates in cell wall construction of the rod shaped bacterium Bacillus subtilis. When growing rapidly, cells lacking ManA exhibit aberrant cell wall architecture, polyploidy and abnormal chromosome morphologies. We demonstrate that these cellular defects are derived from the role played by ManA in cell wall formation. Furthermore, we show that ManA is required for maintaining the proper carbohydrate composition of the cell wall, particularly of teichoic acid constituents. This perturbed cell wall synthesis causes asynchrony between cell wall elongation, division and nucleoid segregation.

Q11 peptide nanofibers are used as a biomaterial for applications such as antigen presentation and tissue engineering, yet detailed knowledge of molecular-level structure has not been reported. The Q11 peptide sequence was designed using heuristics-based patterning of hydrophobic and polar amino acids with oppositely charged amino acids placed at opposite ends of the sequence to promote antiparallel β-sheet formation. In this work, we employed solid-state nuclear magnetic resonance spectroscopy (NMR) to evaluate whether the molecular organization within Q11 self-assembled peptide nanofibers is consistent with the expectations of the peptide designers. We discovered that Q11 forms a distribution of molecular structures. NMR data from two-dimensional (2D) 13C-13C dipolar-assisted rotational resonance indicate that the K3 and E9 residues between Q11 β-strands are spatially proximate (within ∼0.6 nm). Frequency-selective rotational echo double resonance (fsREDOR) on K3 Nζ and E9 Cδ-labeled sites showed that approximately 9% of the sites are close enough for salt bridge formation to occur. Surprisingly, dipolar recoupling measurements revealed that Q11 peptides do not assemble into antiparallel β-sheets as expected, and structural analysis using Fourier-transform infrared spectroscopy and 2D NMR alone can be misleading. 13C PITHIRDS-CT dipolar recoupling measurements showed that the most abundant structure consists of parallel β-sheets, in contrast to the expected antiparallel β-sheet structure. Structural heterogeneity was detected from 15N{13C} REDOR measurements, with approximately 22% of β-strands having antiparallel nearest neighbors. We cannot propose a complete structural model of Q11 nanofibers because of the complexity involved when examining structurally heterogeneous samples using NMR. Altogether, our results show that while heuristics-based patterning is effective in promoting β-sheet formation, designing a peptide sequence to form a targeted β-strand arrangement remains challenging.

Several brittle culm (bc) mutants known in grasses are considered excellent materials to study the process of secondary cell wall formation. The brittle phenotype of the rice bc5 (brittle node) mutant appears exclusively in the developed nodes, which is distinct from other bc mutants (bc1, 2, 3, 4, 6 and 7) that show the brittle phenotype in culms and leaves. To address the defects of the rice bc5 mutant in node-specific cell wall formation, we analyzed tissue morphology and cell wall composition. The bc5 mutation was found to affect the cell wall deposition of node sclerenchyma tissues at 1 week after heading, the stage at which the cell wall sugar content is reduced, in the bc5 nodes, compared with wild-type nodes. Moreover, decreased accumulation of lignin and thickness of cell walls in the sclerenchyma tissues were also observed in the bc5 nodes. The amounts of cellulose and hemicellulose were reduced to 53 and 65% of those in the wild-type plants, respectively. Sugar composition and glycosidic linkage analyses of the hemicellulose showed that the accumulation of glucuronosyl arabinoxylan in bc5 nodes was perturbed by the mutation. The bc5 locus was narrowed to an approximately 3.1 Mb region of chromosome 2, where none of the other bc genes is located. The bc5 mutation appeared to reduce the expression levels of the OsCesA genes in the nodes after heading. The results indicate that the BC5 gene regulates the development of secondary cell walls of node sclerenchyma tissues.

The plant secondary cell wall is the major source of lignocellulosic biomass, a renewable energy resource that can be used for bioethanol production. To comprehensively identify transcription factors (TFs), glycosyltransferase (GT) and glycosyl hydrolase (GH) involved in secondary cell wall formation in rice (Oryza sativa), co-expression network analysis was performed using 68 microarray data points for different rice tissues and stages. In addition to rice genes encoding orthologs of Arabidopsis thaliana TFs known to regulate secondary cell wall formation, the network analysis suggested many novel TF genes likely to be involved in cell wall formation. In the accompanying paper (Hirano et al.), several of these TFs are shown to be involved in rice secondary cell wall formation. Based on a comparison of the rice and Arabidopsis networks, TFs were classified as common to both species or specific to each plant species, suggesting that in addition to a common transcriptional regulatory mechanism of cell wall formation, the two plants may also use species-specific groups of TFs during secondary wall formation. Similarly, genes encoding GT and GH were also classified as genes showing species-common or species-specific expression patterns. In addition, genes for primary or secondary cell wall formation were also suggested. The list of rice TF, GT and GH genes provides an opportunity to unveil the regulation of secondary cell wall formation in grasses, leading to optimization of the cell wall for biofuel production.

Arabidopsis thaliana transcription factors belonging to the ERFIIId and ERFIIIe subclade (ERFIIId/e) of the APETALA 2/ethylene response factor (AP2/ERF) family enhance primary cell wall (PCW) formation. These transcription factors activate expression of genes encoding PCW-type cellulose synthase (CESA) subunits and other genes for PCW biosynthesis. In this study, we show that fiber-specific expression of ERF035-VP16 and ERF041-VP16, which are VP16-fused proteins of ERFIIId/e members, promote cell wall thickening in a wild-type background with a concomitant increase of alcohol insoluble residues (cell wall content) per fresh weight (FW) and monosaccharides related to the PCW without affecting plant growth. Furthermore, in the ERF041-VP16 lines, the total amount of lignin and the syringyl (S)/guaiacyl (G) ratio decreased, and the enzymatic saccharification yield of glucose from cellulose per fresh weight improved. In these lines, PCW-type CESA genes were upregulated and ferulate 5-hydropxylase1 (F5H1), which is necessary for production of the S unit lignin, was downregulated. In addition, various changes in the expression levels of transcription factors regulating secondary cell wall (SCW) formation were observed. In conclusion, fiber cell-specific ERF041-VP16 improves biomass yield, increases PCW components, and alters lignin composition and deposition and may be suitable for use in future molecular breeding programs of biomass crops.

Pathogenic bacteria in human system mature through the bio-synthesis of protective layer known as cell wall. This bacterial cell wall growth occurs in the presence of enzyme released by it. After maturation by the cell wall formation, pathogenic bacteria become harmful for human body as they are responsible for different diseases. Antibiotics or drugs are employed for curing bacterial diseases through the inhibition of this maturation process and it occurs by the binding progression of antibiotics with the released enzyme. But nowadays, drugs or antibiotics like [Formula: see text]-lactum family (Amoxcillin) which are generally used for inhibition of bio-synthesis of cell wall become ineffective due to evolution of antibiotic resistance by the bacteria. Antibiotic resistance occurs when an antibiotic has lost its ability to effectively control or kill bacterial growth. As a result, the bacteria becomes “resistant” and continue to multiply for the generation of robust pathogenic bacteria in spite of drug administration. This is due to the release of another type of enzyme by the resistant bacteria which binds with the active antibiotic or drug making it ineffective. Hence, another type of drug (Clauvanic acid) is combined to resist the activity of drug hydrolyzing enzyme so that the initial drug can act effectively. Hence a combination of drug therapy is applied to cure the bacterial diseases successfully. We developed a mathematical model based on the bacterial enzyme and bacterial cell wall proliferation mechanism and showed how we can reduce the bacterial infection in the resistant cases with application of combination drugs (Amoxcillin and Clauvanic acid) to restore normal health. Based on the enzymatic activity and individual drug dynamics we studied the overall system under the single drug and combinational drug administration through our formulated model analysis. We also demonstrated the different dosing time interval and dosing concentration to evaluate the optimized drug administration for arresting the cell wall formation completely. Sensitivity of the different kinetic rate constant also has been performed with subject to drug hydrolyzing enzyme. Our analytical and numerical studies also confirm the efficiency of the combinational drug treatment compared to single drug treatment being more effective in drug resistant cases providing recovery from bacterial disease.

Protoplasts of Saccharomyces cerevisiae incubated in regenerating conditions secrete cell wall components in order to allow the biosynthesis of this structure. During the first hours of incubation, many of these are not retained in the forming cell wall but remain in the medium. We have developed a method for collecting the secreted proteins and have analysed these by two-dimensional electrophoresis to obtain a reference map of putative cell wall proteins. Several proteins were identified by microsequencing or immunoblotting; namely, cell wall hydrolytic enzymes, heat shock proteins, glycolytic enzymes and others. Some beta-1,3- and beta-1, 6-glucosylation was detected in the proteins secreted by regenerating protoplasts.

Primary plant cell walls contain highly hydrated biopolymer networks, whose major chemistry is known but whose relationship to architectural and mechanical properties is poorly understood. Nuclear magnetic resonance spectroscopy has been used to characterize segmental mobilities via relaxation and anisotropy effects in order to add a dynamic element to emerging models for cell wall architecture. For hydrated onion cell wall material, single pulse excitation revealed galactan (pectin side chains), provided that dipolar decoupling was used, and some of the pectin backbone in the additional presence of magic angle spinning. Cross-polarization excitation revealed the remaining pectin backbones, which exhibited greater mobility (contact time dependence, dipolar dephasing) than the cellulose component, whose noncrystalline and crystalline fractions showed no mobility discrimination. 1HT2 behavior could be quantitatively interpreted in terms of high resolution observabilities. Mobility-resolved spectroscopy of cell walls from tomato fruit, pea stem, and tobacco leaf showed similar general effects. Nuclear magnetic resonance study of the sequential chemical extraction of onion cell wall material suggests that galactans fill many of the network pores, that extractability of pectins is not dependent on segmental mobility, and that some pectic backbone (and not side chain) is strongly associated with cellulose. Analysis of the state of cellulose in four hydrated cell walls suggests a noncrystalline content of 60–80% and comparable amounts of Iα and Iβ polymorphs in the crystalline fraction. Comparison with micrographs for onion cell walls shows that noncrystalline cellulose does not equate to chains on fibril surfaces, and chemical shifts show that fully solvated cellulose is not a significant component in cell walls. © 1996 John Wiley & Sons, Inc.

The conformation of d-alanine (d-Ala) groups of bacterial teichoic acid is a central, yet untested, paradigm of microbiology. The d-Ala binds via the C-terminus, thereby allowing the amine to exist as a free cationic NH(3)(+) group with the ability to form a contact ion pair with the nearby anionic phosphate group. This conformation hinders metal chelation by the phosphate because the zwitterion pair is charge neutral. To the contrary, the repulsion of cationic antimicrobial peptides (CAMPs) is attributed to the presence of the d-Ala cation; thus the ion pair does not form in this model. Solid-state nuclear magnetic resonance (NMR) spectroscopy has been used to measure the distance between amine and phosphate groups within cell wall fragments of Bacillus subtilis. The bacteria were grown on media containing (15)N d-Ala and beta-chloroalanine racemase inhibitor. The rotational-echo double-resonance (REDOR) pulse sequence was used to measure the internuclear dipolar coupling, and the results demonstrate (1) the metal-free amine-to-phosphate distance is 4.4 A and (2) the amine-to-phosphate distance increases to 5.4 A in the presence of Mg(2+) ions. As a result, the zwitterion exists in a nitrogen-oxygen ion pair configuration providing teichoic acid with a positive charge to repel CAMPs. Additionally, the amine of d-Ala does not prevent magnesium chelation in contradiction to the prevailing view of teichoic acids in metal binding. Thus, the NMR-based description of teichoic acid structure resolves the contradictory models, advances the basic understanding of cell wall biochemistry, and provides possible insight into the creation of new antibiotic therapies.

Cellular Characteristics of Dinitroaniline Herbicide-Resistant Goosegrass (<i>Eleusine indica</i>)