Nous tenons à remercier Andrew Carroll, Bright Chaibang, Purbasha Sarkar (Energy Biosciences Institute, Berkeley), Bahram Parvin (Lawrence Berkeley National Laboratory) et Vincent L. Chiang (North Carolina State University) pour des collaborations fructueuses et des discussions utiles. Ce travail a été soutenu par l&#39;Institut de l&#39;énergie Biosciences. Le travail à la Fonderie Moléculaire a été soutenue par l&#39;Office of Science, Bureau des sciences fondamentales de l&#39;énergie, du Département américain de l&#39;Énergie en vertu du contrat n ° DE-AC02-05CH1123.

1. Préparation des échantillons <ol><li> Montez les échantillons végétaux hydratée, de bois de peuplier, par exemple la tige ou une tige d&#39;Arabidopsis thaliana, dans le microtome. </li><li> Couper de fines sections (typiquement 20 um d&#39;épaisseur) à partir du tissu natif. </li><li> Transfert de la section centrale sur une lame de microscope en verre. </li><li> Faire tremper la section centrale dans D 2 O et couvrir avec une lamelle de verre, qui est scellé sur la lame du microscope pour empêcher l&#39;évaporation de D 2 O. La section centrale est maintenant prêt pour l&#39;imagerie ou il peut être stocké pour une utilisation future. </li></ol> 2. Mesure des échantillons <ol><li> Appliquer l&#39;huile d&#39;immersion à l&#39;objectif du microscope et / ou de la lamelle. </li><li> Lieu et sécuriser la lame de microscope sur la scène de balayage piézo-électriques du microscope, avec la lamelle face à l&#39;objectif du microscope. </li><li> Voir l&#39;échantillon à travers la lamelle en utilisant un microscope à haute objectif chiffré d&#39;ouverture d&#39;immersion (100x, NA = 1,40) et de localiser la zone d&#39;échantillon d&#39;intérêt. </li><li> Après avoir éteint toutes les autres sources lumineuses de laboratoire et d&#39;un microscope, la position résolue de mesures sont effectuées par microspectroscopie concentrant passe-bande-filtrée monochromatique vert clair (λ = 532 nm) à partir d&#39;un cw-laser sur l&#39;échantillon avec une puissance typique de 10 à 30 mW ( voir la Figure 1 pour un schéma de l&#39;installation). Autofluorescence peut se produire dans certains échantillons, ce qui peut interdire des mesures utiles, dans laquelle l&#39;excitation cas de la lumière laser de longueur d&#39;onde plus peut être souhaitable. </li><li> L&#39;arrière-lumière diffusée Raman Stokes décalée est collectée par l&#39;objectif du microscope, passe à travers un miroir dichroïque, un trou d&#39;épingle, qui sert de filtre spatial dans la configuration confocale, et un filtre longpass, et se concentre dans la fente d&#39;un réseau spectromètre, où la lumière est dispersée spectralement et détecté par une caméra CCD refroidie, ce qui donne un spectre Raman. Un spectre Raman du bois de peuplier est montré dans la figure 2, avec des bandes de lignine caractéristique dans la région spectrale entre 1600 et 1700 cm -1. </li><li> Pour l&#39;imagerie chimique et la visualisation de la distribution spatiale de lignine, une carte à deux dimensions spectrales est acquis par trame de balayage de l&#39;échantillon à travers la focalisation du laser avec l&#39;étape de balayage piézo-électrique et l&#39;enregistrement d&#39;un spectre Raman pour chaque position de l&#39;échantillon. Cartes en trois dimensions spectrales peuvent être générées par l&#39;empilement de cartes en deux dimensions pour lequel la focalisation du laser a été intensifiée consécutivement le long de la direction z. </li></ol> 3. Analyse des données <ol><li> Pour Imagerie chimique et la visualisation de lignine, les données recueillies sont analysées à l&#39;aide de MATLAB (The MathWorks, version 7.7). Les données sont organisées dans un cube en trois dimensions hyperspectrale, qui est composé de deux dimensions spatiales et une troisième dimension spectrale des signaux. </li><li> Pour l&#39;analyse de la lignine, la région spectrale entre 1550 et 1700 cm -1 est considéré (voir figure 2). La répartition spatiale de la lignine est visualisée par l&#39;intégration de l&#39;intensité de 1550 à 1700 cm -1 des spectres de référence corrigée (voir figure 3). Comme une alternative à la correction de base, dérivée seconde spectres peuvent être calculées et les sommets de deuxième dérivés utilisés pour l&#39;analyse. </li><li> Localisation lignine et de la chimie, notamment en matière de coniferaldehyde et des groupements alcool coniférylique, peut encore être analysée en évaluant la surface sous les pics gaussiens équipé des trois bandes trouvé entre 1600 et 1700 cm-1 (voir encart de la figure 2 et Refs 15. - 17). </li><li> La normalisation de l&#39;intensité entre les différentes cartes spectrale est réalisée en utilisant comme référence la hauteur du pic de la bande qui s&#39;étend autour extrinsèques OD 2500 cm -1 dans les spectres de lumière moyenne, qui sont obtenus par la classification des k-means clustering. Cela est essentiel et permet de comparer les intensités du signal entre les différentes mesures de lignine, de tissus, d&#39;échantillons et d&#39;espèces. </li></ol> 4. Les résultats représentatifs Un spectre Raman représentatives du bois de peuplier tige (Populus angustifolia) est montré dans la figure 2. Bandes de lignine caractéristiques se trouvent dans la région spectrale entre 1600 et 1700 cm -1. À titre d&#39;exemple, la distribution spatiale de la lignine dans un bois de peuplier section est présentée en figure 3. Par rapport à l&#39;image visible, morphologiquement distinctes régions paroi cellulaire devient clairement identifiable en raison de l&#39;intensité du signal lignine différente. Haute intensité du signal lignine est observée dans les coins de cellule (CC) et, un peu moins, dans la lamelle moyenne composé (LMC). Des montants inférieurs, mais non négligeable, de lignine sont observées dans la couche murale S2 des fibres. La variabilité de l&#39;intensité du signal lignine se trouve dans une certaine mesure au sein du CC, la LMC et S2, en particulier de fibre en fibre. La résolution latérale spatiale dans nos mesures est d&#39;environ 300 nm. La qualité des données se prête bien à comparer les lignification entre leséchantillons et de disséquer la lignine de chimie 15. <img src="/files/ftp_upload/2064/2064fig1.jpg" alt="Figure 1" /> Figure 1: Un schéma de la configuration instrumentale BP:. Filtre passe-bande; DM: miroir dichroïque, PH: sténopé; LP: longpass filtre. <img src="/files/ftp_upload/2064/2064fig2.jpg" alt="Figure 2" /> Figure 2: Un spectre Raman représentatives du bois de peuplier tige (Populus angustifolia) enregistrées dans D 2 O. La zone en surbrillance spectrale (voir aussi l&#39;encadré) marque la région spectrale ayant trois sommets spécifiquement attribuables à la lignine. <img src="/files/ftp_upload/2064/2064fig3.jpg" alt="Figure 3" /> Figure 3: Raman lignine image (en bas) d&#39;un bois de peuplier de section (en haut: image visible), obtenue en intégrant l&#39;intensité du signal Raman de 1550 à 1700 cm -1.

<ol>
	<li>Herrera, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bonkers+about+biofuels.">Bonkers about biofuels.</a> Nat Biotechnol. 24, 755-760 (2006).</li><li>Himmel, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomass+recalcitrance:+Engineering+plants+and+enzymes+for+biofuels+production.">Biomass recalcitrance: Engineering plants and enzymes for biofuels production.</a> Science. 315, 804-807 (2007).</li><li>Pauly, M., Keegstra, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-wall+carbohydrates+and+their+modification+as+a+resource+for+biofuels.">Cell-wall carbohydrates and their modification as a resource for biofuels.</a> Plant J. 54, 559-568 (2008).</li><li>Pauly, M., Keegstra, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+and+metabolism+'Tear+down+this+wall.">Physiology and metabolism 'Tear down this wall.</a> Curr Opin Plant Biol. 11, 233-235 (2008).</li><li>Ragauskas, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+path+forward+for+biofuels+and+biomaterials.">The path forward for biofuels and biomaterials.</a> Science. 311, 484-489 (2006).</li><li>Somerville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofuels.">Biofuels.</a> Curr Biol. 17, R115-R119 (2007).</li><li>Ralph, J., Brunow, G., Boerjan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lignins in Encyclopedia of Life Sciences. , (2007).</li><li>Chiang, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+rags+to+riches.">From rags to riches.</a> Nat Biotechnol. 20, 557-558 (2002).</li><li>Atalla, R. H., Agarwal, U. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raman+microprobe+evidence+for+lignin+orientation+in+the+cell+walls+of+native+woody+tissue.">Raman microprobe evidence for lignin orientation in the cell walls of native woody tissue.</a> Science. 227, 636-638 (1985).</li><li>Atalla, R. H., Agarwal, U. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recording+Raman+spectra+from+plant+cell+walls.">Recording Raman spectra from plant cell walls.</a> J Raman Spectrosc. 17, 229-231 (1986).</li><li>Fukushima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+syringyl+to+guaiacyl+ratio+in+lignin+biosynthesis.">Regulation of syringyl to guaiacyl ratio in lignin biosynthesis.</a> J Plant Res. 114, 499-508 (2001).</li><li>Agarwal, U. 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Matières lignocellulosiques sont hiérarchiques et hétérogènes en ce qui concerne à la fois à structure et la composition. Pour un des outils de caractérisation en profondeur d&#39;analyse qui ont une sensibilité chimique, la résolution spatiale, et qui donnent un aperçu de ces matériaux dans le contexte natif sont souhaitables. La méthode décrite permet la visualisation de la lignine et la comparaison de la lignification de la biomasse végétale lignocellulosique avec une résolution spatiale qui est sous...

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label-free in situ imaging of lignification in plant cell walls

Réunion de l&#39;énergie demande croissante en toute sécurité et est efficace une urgence planétaire. Par conséquent, la recherche sur la production de biocarburants, qui vise à trouver des solutions rentables et durables est devenue une tâche d&#39;actualité et critique. La biomasse lignocellulosique est en passe de devenir la principale source de biomasse pour la conversion en biocarburants liquides 1-6. Cependant, la réticence de ces matières végétales paroi cellulaire à la dégradation rentable et efficace présente un obstacle majeur pour leur utilisation dans la production de biocarburants et de produits chimiques 4. En particulier, la lignine, un complexe et irrégulière poly-phénylpropanoïde hétéropolymère, devient problématique à l&#39;après-récolte déconstruction de la biomasse lignocellulosique. Par exemple dans la conversion de la biomasse pour les biocarburants, il inhibe la saccharification dans les processus visant à produire des sucres simples pour la fermentation 7. L&#39;utilisation efficace de la biomasse végétale à des fins industrielles est en effet largement tributaire de la mesure dans laquelle la paroi cellulaire végétale est lignifiée. L&#39;élimination de la lignine est un facteur limitant coûteux et 8 et de la lignine est donc devenu une clé de l&#39;amélioration des plantes et de cibler le génie génétique en vue d&#39;améliorer la conversion paroi cellulaire. Des outils analytiques qui permettent la caractérisation précise rapide de lignification des parois cellulaires des plantes de plus en plus important pour évaluer un grand nombre de populations d&#39;oiseaux nicheurs. Procédures d&#39;extraction pour l&#39;isolement des composants natifs tels que la lignine sont inévitablement destructeur, entraînant chimiques importantes et des modifications structurelles 9-11. Analyse chimique des méthodes in situ sont donc des outils précieux pour la caractérisation de la composition et la structure de matériaux lignocellulosiques. Microscopie Raman est une technique qui s&#39;appuie sur la diffusion inélastique ou Raman de la lumière monochromatique, comme celle d&#39;un laser, lorsque l&#39;évolution de l&#39;énergie des photons laser est liée à des vibrations moléculaires et présente une valeur intrinsèque sans étiquette moléculaire &quot;empreinte digitale&quot; de l&#39;échantillon . Microscopie Raman peut se permettre des mesures non destructives et relativement bon marché avec une préparation minimale de l&#39;échantillon, ce qui donne un aperçu de la composition chimique et la structure moléculaire dans un proche état natif. Imagerie chimique par microscopie confocale Raman a été précédemment utilisé pour la visualisation de la distribution spatiale de la cellulose et la lignine dans les parois cellulaires du bois 12-14. Basé sur ces résultats antérieurs, nous avons récemment adopté cette méthode pour comparer la lignification de type sauvage et de la lignine déficientes transgéniques Populus trichocarpa (peuplier) tige de bois 15. L&#39;analyse des bandes Raman lignine 16,17 dans la région spectrale entre 1600 et 1700 cm -1, l&#39;intensité du signal de lignine et de localisation ont été cartographiés in situ. Notre approche visualisés différences de teneur en lignine, la localisation et la composition chimique. Plus récemment, nous avons démontré l&#39;imagerie Raman de polymères paroi cellulaire chez Arabidopsis thaliana avec une résolution latérale qui est sous-um 18. Ici, cette méthode est présenté offrant une visualisation de la lignine dans les parois des cellules végétales et la comparaison de la lignification dans les différents tissus, d&#39;échantillons ou d&#39;espèces, sans coloration ou l&#39;étiquetage des tissus.

Watch this Scientific Journal Video about Label-free in situ Imaging of Lignification in Plant Cell Walls at JoVE.com

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

Une méthode basée sur la microscopie confocale Raman est présenté qui offre sans étiquette de visualisation de la lignine dans les parois des cellules végétales et la comparaison de la lignification dans les différents tissus, d&#39;échantillons ou d&#39;espèces.

Sans étiquette In situ</emImagerie> de lignification dans les parois des cellules végétales

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

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Biology

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

12.3K vues.  University of California, Berkeley. Une méthode basée sur la microscopie confocale Raman est présenté qui offre sans étiquette de visualisation de la lignine dans les parois des cellules végétales et la comparaison de la lignification dans les différents tissus, d'échantillons ou d'espèces.

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

In situ Imaging of the Mouse Thymus Using 2-Photon Microscopy

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development. To follow the migration of individuals in a crowd of thymocytes , we generate neonatal chimeras where less than one percent of the thymocytes are derived from a donor that is transgenic for a ubiquitously express fluorescent protein. To generate these partial hematopoetic chimeras, neonatal recipients are injected with bone marrow between 3-7 days of age. After 4-6 weeks, the mouse is sacrificed and the thymus is carefully dissected and bissected preserving the architecture of the tissue that will be imaged. The thymus is glued onto a coverslip in preparation for ex vivo imaging by TPM. During imaging the thymus is kept in DMEM without phenol red that is perfused with 95% oxygen and 5% carbon dioxide and warmed to 37&deg;C. Using this approach, we can study the events required for the generation of a diverse T cell repertoire.

We present step-by-step instructions for the generation of neonatal chimeras as well as the dissection and preparation of the thymus for ex vivo imaging by 2-Photon Microscopy.

Dans l&#39;imagerie in situ de la souris en utilisant deux Thymus microscopie biphotonique

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development.  To follow the ...

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Biologie

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.

Partie analyse complète de la composition des parois des cellules végétales (biomasse lignocellulosique) I: La lignine

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 ...

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.

Analyse de la composition globale de la paroi cellulaire végétale (biomasse lignocellulosique) Partie II: Glucides

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

Changement de forme d&#39;imagerie cellulaire en vie Drosophile Embryons

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

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.

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.

Profilage Glycan des polymères végétaux de la paroi cellulaire en utilisant 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 ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Vivre imagerie de dédouanement cellulaire par apoptose au cours<em&gt; Drosophila</em&gt; Embryogenèse

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Résolution Quantification haut de Crystalline Cellulose Accumulation dans<em&gt; Arabidopsis</em&gt; Roots pour surveiller cellulaire spécifique d&#39;un tissu mural Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Hybrid-Cut: Une méthode Sectionnement améliorée pour les échantillons de tissus végétaux récalcitrantes

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

Identification de nouveaux régulateurs de la transpiration végétale par le dépistage thermique à grande échelle à Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits obesity and many associated diseases including cardiovascular disorders, type II diabetes, and cancer. To advance the current understanding of lipid metabolic regulation, quantitative methods to precisely measure in vivo lipid storage levels in time and space have become increasingly important and useful. Traditional approaches to analyze lipid storage are semi-quantitative for microscopic assessment or lacking spatio-temporal information for biochemical measurement. Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology that enables rapid and quantitative detection of lipids in live cells with a subcellular resolution. As the contrast is exploited from intrinsic molecular vibrations, SRS microscopy also permits four-dimensional tracking of lipids in live animals. In the last decade, SRS microscopy has been widely used for small molecule imaging in biomedical research and overcome the major limitations of conventional fluorescent staining and lipid extraction methods. In the laboratory, we have combined SRS microscopy with the genetic and biochemical tools available to the powerful model organism, Caenorhabditis elegans, to investigate the distribution and heterogeneity of lipid droplets across different cells and tissues and ultimately to discover novel conserved signaling pathways that modulate lipid metabolism. Here, we present the working principles and the detailed setup of the SRS microscope and provide methods for its use in quantifying lipid storage at distinct developmental timepoints of wild-type and insulin signaling deficient mutant C. elegans.

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows selective, label-free imaging of specific chemical moieties and it has been effectively employed to image lipid molecules in vivo. Here, we provide a brief introduction to the principle of SRS microscopy and describe methods for its use in imaging lipid storage in Caenorhabditis elegans.

Imagerie sans étiquette de la dynamique de stockage des lipides chez Caenorhabditis elegans à l’aide de la microscopie de diffusion Raman stimulée

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits ...

Sans étiquette In situ de lignification dans les parois des cellules végétales