우리는 재정 지원을 위해 중국 과학 기술부 (2016YDF0600803)에 감사드립니다.

1. 샘플 준비 <ol><li> 식물 샘플 ( 예 : 포플러 줄기)에서 작은 조직 블록 (약 3mm x 3mm x 5mm)을 자릅니다. </li><li> 조직을 끓는 탈 이온수에 30 분간 담근다. 즉시 실온 (RT)에서 30 분 동안 탈 이온수로 옮긴다. 티슈가 용기 바닥에 가라 앉을 때까지이 단계를 반복하여 조직의 공기가 제거되었고 조직이 부드럽게되었다는 것을 나타냅니다. 참고 :이 단계 이전에 바닥에 가라 앉은 샘플의 경우 일반적으로이주기를 3-5 번 반복하십시오. </li><li> 순수한 PEG뿐만 아니라 탈 이온수에 폴리에틸렌 글리콜 (PEG)의 20, 50, 70, 90 % (v / v) 분취 량을 준비하고 65 ° C에서 용액을 유지하십시오. </li><li> 수분을 치환하고 PEG가 침투하도록 일련의 등급이 매겨진 PEG 조에 조직을 배양합니다. 참고 : 일반적으로 분자량 2,000 (PEG2000) 인 PEG가 삽입에 사용됩니다. <ol><li> 조직을 g로 처리하십시오.(a) 1 시간 동안 20 % PEG, (b) 1.5 시간 동안 50 % PEG, (c) 2 시간 동안 70 % PEG, (d) 2 시간 동안 90 % PEG, 및 (e) 10 시간 동안 100 % PEG. </li></ol></li><li> 카세트를 오븐에서 65 ° C로 예열하십시오. 블록이 들어있는 PEG를 카세트에 넣은 다음 예열 된 핀셋 또는 바늘을 사용하여 조직 블록을 원하는 위치에 놓습니다. </li><li> 천천히 카세트를 식히고 사용할 때까지 실온에 티슈를 보관하십시오. </li><li> 날카로운 면도날을 사용하여 작은 블록 (약 1cm x 1cm x 2cm)에 표적 조직이 들어있는 PEG 블록을 해부하고 그것을 마이크로톰에 올려 놓습니다. </li><li> PEG 블록 (일반적으로 3-10 μm)에서 얇은 절편을 잘라냅니다. </li><li> 시계에서 10 번 탈 이온수로 섹션을 씻어 조직에서 PEG를 제거합니다. </li><li> 추출물을 제거하기 위해 6 시간 동안 톨루엔 / 에탄올 (2 : 1, v / v)로 섹션을 담그십시오. 탈 이온수 65 mL, 아세트산 0.5 mL 및 나트륨 클로라이드 0.6 g을 혼합하여 반응 액을 조제한다.비커에 의식. 5 mL 바이알에 한 개의 섹션과 3 mL의 반응 액을 첨가한다. 유리 병의 상단에 나사를 조이십시오. </li><li> 유리 병을 75oC에서 2 시간 가열하여 조직의 리그닌을 제거한다. 시계 유리에있는 탈 이온수로 섹션을 10 번 헹굽니다. </li><li> 미처리 / 탈 리그닌 부분을 유리 현미경 슬라이드로 옮긴다. 브러시 또는 바늘을 사용하여 조심스럽게 섹션을 펼칩니다. 티슈 페이퍼로 여분의 탈 이온수를 제거하십시오. </li><li> 섹션을 D 2 O에 담그십시오. 샘플을 유리 커버 슬립으로 덮으십시오. D 2 O의 증발을 막기 위해 매니큐어로 커버 슬립을 밀봉하십시오. </li></ol> 2. 스펙트럼 획득 <ol><li> 공 촛점 라만 현미경의 장비 운영 소프트웨어를 엽니 다. 레이저 (파장 = 532 nm)를 켜고 100 배 현미경 대물 렌즈를 사용하여 결정 실리콘 표면에 초점을 맞추고 보정 버튼을 클릭하여 장비를 보정합니다. </li><li> 계기 스위치광학 현미경 모드로 설정하고 현미경 램프를 켭니다. 커버 슬립이 목표를 향하도록 스테이지에 현미경 슬라이드를 장착하십시오. </li><li> 20X 현미경 대물 렌즈로 샘플을보고 관심 영역을 찾습니다. 커버 슬립에 침지 오일을 바르고 액침 현미경 대물 렌즈 (60X, 개구 수 NA = 1.35)로 전환합니다. 샘플 표면에 초점을 맞 춥니 다. </li><li> 장비를 라만 테스트 모드로 전환하고 현미경 램프를 끕니다. </li><li> 직사각형 도구를 사용하여 매핑 영역을 선택하십시오. 단계 크기를 변경하여 얻은 스펙트럼의 수를 결정합니다. 주 : 스텝 크기 (일반적으로 대물 렌즈의 개구 수에 의해 계산 된 스폿 직경보다 큰, 이론적으로는 1.22λ / NA)보다 유의하십시오. 이 크기보다 작은 경우 오버 샘플링이 발생합니다. </li><li> 최적의 스펙트럼 파라미터를 설정하여 적절한 획득 시간 (일반적으로 8 시간 미만)에서 최적의 신호 대 잡음비 (SNR) 및 스펙트럼 품질을 얻고,샘플 적합성에 관한 정보. 일반적으로 레이저 (532 nm), 필터 (100 %), 구멍 (300), 슬릿 (100), 분광계 (1,840 cm -1 ), 홈 (1200 t), 대물 렌즈 60X 오일) 및 수집 시간 (2 초). </li><li> 데이터 처리 전에 스펙트럼 데이터를 저장하고 범용 형식 ( 예 : TXT 파일)으로 변환하십시오. </li></ol> 3. 데이터 분석 <ol><li> 스펙트럼 데이터 (TXT 파일)를 데이터 분석 소프트웨어 ( 예 : Matlab)에로드하십시오. 데이터 세트에 노이즈 감소 기술을 적용하여 SNR을 향상시킵니다 ( 예 : Savitzsky-Golay 알고리즘 또는 웨이블릿 알고리즘) </li><li> 미처리 샘플을 사용하여 리그닌과 다당류의 이미지를 생성하십시오. 리그닌 영상의 경우, 방향족 링 대칭 스트레칭 진동 때문에 1,600 cm -1 부근의 스펙트럼 피크를 고려하십시오. 다당류 이미징 (셀룰로오스 및 헤미 셀룰로오스 포함)의 경우 스펙트럼 피크 aCH와 CH 2 뻗기 때문에 2,889 cm -1 라운드. </li><li> delignified 샘플을 사용하여 셀룰로오스와 헤미 셀룰로스의 이미지를 생성합니다. 획득 된 스펙트럼에서 자기 모델링 곡선 분해능 (SMCR)을 수행하여 셀룰로오스와 헤미 셀룰로스의 스펙트럼을 구별하고 그 분포를 영상화하십시오. </li><li> 획득 한 데이터에 대해 주성분 분석 (PCA) 및 클러스터링 분석을 수행하여 다른 셀 벽 레이어의 라만 스펙트럼을 구별합니다. </li></ol>

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저자는 공개 할 것이 없습니다.

식물 세포 벽은 세포 모서리 (CC), 2 차벽 (S1, S2 및 S3 층을 갖는 SW) 및 복합 중간 엽 줄기 (CML, 중층 및 인접한 주 엽산)를 포함하는 여러 층으로 조직화 된 복합체이다. 벽), 이는 샘플 준비 동안 평평한 표면을 얻는 것을 어렵게 만듭니다. 따라서 나무보다 복잡한 구조를 가진 식물 샘플, 특히 잔디는 미세 절단을 위해 응고 될 필요가 있습니다. PEG는 물에 녹기 때문에 절단 및 라만 조사를위한 이상적...

Plant biomass is the most abundant renewable resource on Earth; is mainly composed of lignin, cellulose, and hemicellulose; and is considered an attractive source of bioenergy and bio-based chemicals1. Unfortunately, it can resist degradation and confer hydrolytic stability or structural robustness to the plant cell wall. Such resistance is attributable to the accessible surface area, biomass particle size, degree of polymerization, cellulose crystallinity, and protective lignin2. A comprehensive understanding of the structural and chemical nature of the plant cell wall is thus significant from the viewpoint of plant biology and chemistry, as well as from that of commercial utilization. Commonly used wet chemistry analyses, such as chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy, only provide average compositional data of the measured sample. Furthermore, these methods are invasive and destroy the original structure of the plant tissue3. 
The Raman imaging technique is a powerful tool for the nondestructive visualization of spatially resolved chemical information4. It uses a laser light to cause inelastic scattering with a photon and relies on changes in polarizability arising from the molecular vibrations. In this case, water causes weak Raman scattering, which makes this approach suitable for in situ investigations of biological samples5. The application of the Raman imaging technique to the plant cell wall can elucidate the structure and composition of plant cell walls in their native state, with the resolution on the scale of the single cell and even of the cell wall layers6. A typical Raman imaging analysis of a plant cell wall generally consists of three steps: 1) sample preparation, 2) spectral acquisition, and 3) data processing. 
Although one of the major advantages of Raman imaging is the ability to achieve label-free and non-destructive spectra with minimal sample preparation, physical sample sectioning is still necessary to expose the surface of interest. This process should be performed carefully to obtain a flat surface, since the technique depends on maintaining optical focus7. Spectral acquisition requires a balance between image quality and extensive acquisition times8. Data processing aims to effectively extract the chemical information from the image data, especially for the components with similar chemical structures, such as cellulose and hemicellulose. Due to the strong spectral overlap, the exact spectra are difficult to discern. In this case, multivariate analysis is a straightforward approach to effectively uncover the hiding structural and chemical information9. This work presents a general protocol describing the use of Raman imaging to visualize the main components in plant cell walls, including lignin, cellulose, and hemicellulose.

<table><tbody><tr><td>Microtome</td><td>Thermo Scientific</td><td>Microm HM430</td><td></td></tr><tr><td>Confocal Raman microscope</td><td>Horiba Jobin Yvon</td><td>Xplora</td><td></td></tr><tr><td>Oven</td><td>Shanghai ZHICHENG</td><td>ZXFD-A5040</td><td></td></tr></tbody></table>

combining raman imaging and multivariate analysis to visualize lignin, cellulose, and hemicellulose in the plant cell wall

식물체 바이오 매스에 라만 이미징을 적용하는 것은 수용액에 대한 공간적 및 조성 정보를 제공 할 수 있기 때문에 증가하고 있습니다. 분석은 일반적으로 광범위한 샘플 준비가 필요하지 않습니다. 라벨을 붙이지 않고도 구조적 및 화학적 정보를 얻을 수 있습니다. 그러나 각 라만 이미지에는 수천 개의 스펙트럼이 포함되어 있습니다. 이것은 숨겨진 정보를 추출 할 때 어려움을 낳습니다. 특히 비슷한 화학 구조를 가진 구성 요소의 경우. 이 연구는이 문제를 해결하기 위해 다변량 분석을 도입합니다. 이 프로토콜은 식물 세포벽 내에서 리그닌, 셀룰로오스 및 헤미 셀룰로스를 비롯한 주성분을 시각화하는 일반적인 방법을 수립합니다. 이 프로토콜에서는 시료 준비, 스펙트럼 획득 및 데이터 처리 절차가 설명됩니다. 이는 시료 준비 및 데이터 분석시 운영자의 기술에 크게 의존합니다. 이 접근법을 사용함으로써 비전문 사용자가 라만 조사를 수행하여 high 품질 데이터 및 식물 세포벽 분석에 대한 의미있는 결과.

그림 1 은 식물 세포벽의 라만 이미징을위한 전형적인 마이크로 라만 시스템의 개요를 나타냅니다. 예를 들어, 포플라 ( Populus nigra L.)의 원래 라만 스펙트럼은 상당한 기준선 드리프트와 스파이크를 가지고 있습니다 ( 그림 2a ). 라만 이미징 데이터 세트 (APRI)에 대한 자동 사전 처리 방법을 수행 한 후,이 두 스펙트럼 오?...

Watch this Scientific Journal Video about Combining Raman Imaging and Multivariate Analysis to Visualize Lignin, Cellulose, and Hemicellulose in the Plant Cell Wall at JoVE.com

Combining Raman Imaging and Multivariate Analysis to Visualize Lignin, Cellulose, and Hemicellulose in the Plant Cell Wall

이 프로토콜은 라만 이미징 및 다 변수 분석을 사용하여 식물 세포 벽에서 리그닌, 셀룰로오스 및 헤미 셀룰로오스를 시각화하는 일반적인 방법을 제시합니다.

라만 이미징과 다변량 분석을 결합하여 식물 세포벽에서 리그닌, 셀룰로오스 및 헤미 셀룰로스를 시각화합니다.

The application of Raman imaging to plant biomass is increasing because it can offer spatial and compositional information on aqueous solutions. The ...

combining-raman-imaging-multivariate-analysis-to-visualize-lignin

Universidad de Cartagena UDEC

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Biochemistry

11.8K 조회수.  Beijing Forestry University.  이 프로토콜은 라만 이미징 및 다 변수 분석을 사용하여 식물 세포 벽에서 리그닌, 셀룰로오스 및 헤미 셀룰로오스를 시각화하는 일반적인 방법을 제시합니다. 

비디오: Combining Raman Imaging and Multivariate Analysis to Visualize Lignin, Cellulose, and Hemicellulose in the Plant Cell Wall

High-throughput Screening of Recalcitrance Variations in Lignocellulosic Biomass: Total Lignin, Lignin Monomers, and Enzymatic Sugar Release

The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the use of non-renewable energy sources. In order to identify which plants, out of a diverse pool, have the desired chemical traits for downstream applications, attributes, such as cellulose and lignin content, or monomeric sugar release following an enzymatic saccharification, must be compared. The experimental and data analysis protocols of the standard methods of analysis can be time-consuming, thereby limiting the number of samples that can be measured. High-throughput (HTP) methods alleviate the shortcomings of the standard methods, and permit the rapid screening of available samples to isolate those possessing the desired traits. This study illustrates the HTP sugar release and pyrolysis-molecular beam mass spectrometry pipelines employed at the National Renewable Energy Lab. These pipelines have enabled the efficient assessment of thousands of plants while decreasing experimental time and costs through reductions in labor and consumables.

Plant cell wall structure and chemistry traits are evaluated to identify ideal feedstocks for biofuels and bio-materials. Standard methods have limitations when applied to large data sets. These high-throughput pretreatment, enzyme saccharification, and pyrolysis-molecular beam mass spectrometry methods compare large numbers of biomass samples with decreased experimental time and cost.

총 리그닌, 리그닌 단량체, 및 효소 설탕 자료 : 리그 노 셀룰로오스 바이오 매스의 반항의 변화의 높은 처리량 검열

The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the ...

연구

환경과학

Preparation of Fungal and Plant Materials for Structural Elucidation Using Dynamic Nuclear Polarization Solid-State NMR

This protocol shows how uniformly 13C, 15N-labeled fungal materials can be produced and how these soft materials should be proceeded for solid-state NMR and sensitivity-enhanced DNP experiments. The sample processing procedure of plant biomass is also detailed. This method allows the measurement of a series of 1D and 2D 13C-13C/15N correlations spectra, which enables high-resolution structural elucidation of complex biomaterials in their native state, with minimal perturbation. The isotope-labeling can be examined by quantifying the intensity in 1D spectra and the polarization transfer efficiency in 2D correlation spectra. The success of dynamic nuclear polarization (DNP) sample preparation can be evaluated by the sensitivity enhancement factor. Further experiments examining the structural aspects of the polysaccharides and proteins will lead to a model of the three-dimensional architecture. These methods can be modified and adapted to investigate a wide range of carbohydrate-rich materials, including the natural cell walls of plants, fungi, algae and bacteria, as well as synthesized or designed carbohydrate polymers and their complex with other molecules.

A protocol for preparing 13C,15N-labeled fungal and plant samples for multidimensional solid-state NMR spectroscopy and dynamic nuclear polarization (DNP) investigations is presented.

곰 팡이의 준비 및 동적인 핵 분극 고체 NMR을 사용 하 여 구조 설명에 대 한 식물 재료

This protocol shows how uniformly 13C, 15N-labeled fungal materials can be produced and how these soft materials should be proceeded for solid-state NMR ...

생화학

Visualizing Lignification Dynamics in Plants with Click Chemistry: Dual Labeling is BLISS!

Lignin is one of the most prevalent biopolymers on the planet and a major component of lignocellulosic biomass. This phenolic polymer plays a vital structural and protective role in the development and life of higher plants. Although the intricate mechanisms regulating lignification processes in vivo strongly impact the industrial valorization of many plant-derived products, the scientific community still has a long way to go to decipher them. In a simple three-step workflow, the dual labeling protocol presented herein enables bioimaging studies of actively lignifying zones of plant tissues. The first step consists in the metabolic incorporation of two independent chemical reporters, surrogates of the two native monolignols that give rise to lignin H- and G-units. After incorporation into growing lignin polymers, each reporter is then specifically labeled with its own fluorescent probe via a sequential combination of bioorthogonal SPAAC/CuAAC click reactions. Combined with lignin autofluorescence, this approach leads to the generation of three-color localization maps of lignin within plant cell walls by confocal fluorescence microscopy and provides precise spatial information on the presence or absence of active lignification machinery at the scale of plant tissues, cells and different cell wall layers.

BLISS, a dual labeling protocol for studying lignification dynamics, was developed. Using synthetic monolignol reporters and a sequential combination of SPAAC and CuAAC bioorthogonal click reactions, this methodology paves the way to in-depth analysis of the factors that regulate the biogenesis of lignins in planta.

Lignification 역학 클릭 화학 식물에서 시각화: 행복은 듀얼 라벨!

Lignin is one of the most prevalent biopolymers on the planet and a major component of lignocellulosic biomass. This phenolic polymer plays a vital ...

화학

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis process, which maintains enzymes far from their substrate. Characterization of these complex interactions is thus a challenge in complex substrates such as LB. The method here measures molecular interactions between fluorophore-tagged molecules and native autofluorescent lignin, to be revealed by F&#246;rster resonance energy transfer (FRET). Contrary to FRET measurements in living cells using two exogenous fluorophores, FRET measurements in plants using lignin is not trivial due to its complex autofluorescence. We have developed an original acquisition and analysis pipeline with correlated observation of two complementary properties of fluorescence: fluorescence emission and lifetime. sFLIM (spectral and fluorescent lifetime imaging microscopy) provides the quantification of these interactions with high sensitivity, revealing different interaction levels between biomolecules and lignin.

This protocol describes an original setup combining spectral and fluorescence lifetime measurements to evaluate F&#246;rster resonance energy transfer (FRET) between rhodamine-based fluorescent probes and lignin polymer directly in thick plant sections.

네이티브 오토플루임션을 기반으로 sFLIM에 의한 식물 섹션의 형광 프로브와 리그닌 간의 상호 작용 측정

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part I: Lignin

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation1 and other products such as biocomposite materials7. Plant biomass remains one of the greatest untapped reserves on the planet4. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses (matrix polysaccharides, and the polyphenol lignin6 and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls provide tensile strength to cells and the entire plants, ward off pathogens, and allow water to be transported throughout the plant; in the case of trees up to more the 100 m above ground level. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant4. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerization by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis. In this first part we focus on the analysis of the polyphenol lignin (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its lignin content by acetylbromide solubilization3, and its lignin composition in terms of its syringyl, guaiacyl- and p-hydroxyphenyl units5. The protocol for analyzing the carbohydrates in lignocellulosic biomass including cellulose content and matrix polysaccharide composition is discussed in Part II2.

Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part I: Lignin

Plant biomass is a major carbon-neutral renewable resource that could be used for the production of biofuels. Plant biomass consists mainly of cell walls, a structurally complex composite material termed lignocellulosics. Here we describe a protocol for a comprehensive analysis of the content and composition of the polyphenolic lignin.

식물 세포 벽의 종합 작곡 분석 (리그노셀룰로오스성 바이오 매스) 부 I : 리그닌

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st ...

생물학

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part II: Carbohydrates

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation2 and other products such as biocomposite materials6. Plant biomass remains one of the greatest untapped reserves on the planet4. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses, and the polyphenol lignin5 and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls contribute extensively to the strength and structural integrity of the entire plant. Despite its necessary rigidity, the cell wall is a highly dynamic entity that is metabolically active and plays crucial roles in numerous cell activities such as plant growth and differentiation5. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant4. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerisation by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its monosaccharide composition of the hemicelluloses and other matrix polysaccharides1, and its content of crystalline cellulose7. The protocol for analyzing the lignin components in lignocellulosic biomass is discussed in Part I3.

Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part II: Carbohydrates

Plant biomass is a major carbon-neutral renewable resource that could be used for the production of biofuels. Plant biomass consists mainly of cell walls, a structurally complex composite material termed lignocellulosics. Here we describe a protocol for a comprehensive analysis of the content and composition of wall derived carbohydrates.

식물 세포 벽의 종합 작곡 분석 (리그노셀룰로오스성 바이오 매스) 2 부 : 탄수화물

Ultrafast Lignin Extraction from Unusual Mediterranean Lignocellulosic Residues

Pretreatment is still the most expensive step in lignocellulosic biorefinery processes. It must be made cost-effective by minimizing chemical requirements as well as power and heat consumption and by using environment-friendly solvents. Deep eutectic solvents (DESs) are key, green, and low-cost solvents in sustainable biorefineries. They are transparent mixtures characterized by low freezing points resulting from at least one hydrogen bond donor and one hydrogen bond acceptor. Although DESs are promising solvents, it is necessary to combine them with an economic heating technology, such as microwave irradiation, for competitive profitability. Microwave irradiation is a promising strategy to shorten the heating time and boost fractionation because it can rapidly attain the appropriate temperature. The aim of this study was to develop a one-step, rapid method for biomass fractionation and lignin extraction using a low-cost and biodegradable solvent. 
In this study, a microwave-assisted DES pretreatment was conducted for 60 s at 800 W, using three kinds of DESs. The DES mixtures were facilely prepared from choline chloride (ChCl) and three hydrogen-bond donors (HBDs): a monocarboxylic acid (lactic acid), a dicarboxylic acid (oxalic acid), and urea. This pretreatment was used for biomass fractionation and lignin recovery from marine residues (Posidonia leaves and aegagropile), agri-food byproducts (almond shells and olive pomace), forest residues (pinecones), and perennial lignocellulosic grasses (Stipa tenacissima). Further analyses were conducted to determine the yield, purity, and molecular weight distribution of the recovered lignin. In addition, the effect of DESs on the chemical functional groups in the extracted lignin was determined by Fourier-transform infrared (FTIR) spectroscopy. The results indicate that the ChCl-oxalic acid mixture affords the highest lignin purity and the lowest yield. The present study demonstrates that the DES-microwave process is an ultrafast, efficient, and cost-competitive technology for lignocellulosic biomass fractionation.

Deep eutectic solvent-based, microwave-assisted pretreatment is a green, fast, and efficient process for lignocellulosic fractionation and high-purity lignin recovery.

특이한 지중해 리그놀룰로오스 잔류물에서 초고속 리닌 추출

Pretreatment is still the most expensive step in lignocellulosic biorefinery processes. It must be made cost-effective by minimizing chemical requirements ...

Label-free in situ Imaging of Lignification in Plant Cell Walls

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find cost-effective and sustainable solutions has become a topical and critical task. Lignocellulosic biomass is poised to become the primary source of biomass for the conversion to liquid biofuels1-6. However, the recalcitrance of these plant cell wall materials to cost-effective and efficient degradation presents a major impediment for their use in the production of biofuels and chemicals4. In particular, lignin, a complex and irregular poly-phenylpropanoid heteropolymer, becomes problematic to the postharvest deconstruction of lignocellulosic biomass. For example in biomass conversion for biofuels, it inhibits saccharification in processes aimed at producing simple sugars for fermentation7. The effective use of plant biomass for industrial purposes is in fact largely dependent on the extent to which the plant cell wall is lignified. The removal of lignin is a costly and limiting factor8 and lignin has therefore become a key plant breeding and genetic engineering target in order to improve cell wall conversion.
Analytical tools that permit the accurate rapid characterization of lignification of plant cell walls become increasingly important for evaluating a large number of breeding populations. Extractive procedures for the isolation of native components such as lignin are inevitably destructive, bringing about significant chemical and structural modifications9-11. Analytical chemical in situ methods are thus invaluable tools for the compositional and structural characterization of lignocellulosic materials. Raman microscopy is a technique that relies on inelastic or Raman scattering of monochromatic light, like that from a laser, where the shift in energy of the laser photons is related to molecular vibrations and presents an intrinsic label-free molecular "fingerprint" of the sample. Raman microscopy can afford non-destructive and comparatively inexpensive measurements with minimal sample preparation, giving insights into chemical composition and molecular structure in a close to native state. Chemical imaging by confocal Raman microscopy has been previously used for the visualization of the spatial distribution of cellulose and lignin in wood cell walls12-14. Based on these earlier results, we have recently adopted this method to compare lignification in wild type and lignin-deficient transgenic Populus trichocarpa (black cottonwood) stem wood15. Analyzing the lignin Raman bands16,17 in the spectral region between 1,600 and 1,700 cm-1, lignin signal intensity and localization were mapped in situ. Our approach visualized differences in lignin content, localization, and chemical composition. Most recently, we demonstrated Raman imaging of cell wall polymers in Arabidopsis thaliana with lateral resolution that is sub-&mu;m18. Here, this method is presented affording visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species without staining or labeling of the tissues.

Label-free in situ Imaging of Lignification in Plant Cell Walls

A method based on confocal Raman microscopy is presented that affords label-free visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species.

라벨 - 무료 현장에서</em식물 세포 벽 Lignification의> 이미징

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays 

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Microarrays를 사용하여 식물 세포의 벽 폴리머의 Glycan 프로파일

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

의 조직 화학 염색<em&gt; 애기 장대</em&gt; 차 전지 벽 요소

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

라만 이미징과 다변량 분석을 결합하여 식물 세포벽에서 리그닌, 셀룰로오스 및 헤미 셀룰로스를 시각화합니다.

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