Siamo grati a Sabin Russell per l&#39;assistenza editing. Questo lavoro è stato parte del DOE Joint BioEnergy Institute (http://www.jbei.org), sostenuto dal Dipartimento dell&#39;Energia degli Stati Uniti, Office of Science, Ufficio di Ricerca Biologica e Ambientale, attraverso il contratto di DE-AC02-05CH11231 tra il Lawrence Berkeley National Laboratory e il Dipartimento dell&#39;Energia degli Stati Uniti.

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+in+the+interaction+of+direct+dyes+with+polysaccharides.">Specificity in the interaction of direct dyes with polysaccharides.</a> Carbohydrate Research. 85, 271-287 (1980).</li><li>Hughes, J., McCully, 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=The+use+of+an+optical+brightener+in+the+study+of+plant+structure.">The use of an optical brightener in the study of plant structure.</a> Stain technology. 50, 319-329 (1975).</li><li>Verbelen, J. P., Kerstens, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarization+confocal+microscopy+and+congo+red+fluorescence:+a+simple+and+rapid+method+to+determine+the+mean+cellulose+fibril+orientation+in+plants.">Polarization confocal microscopy and congo red fluorescence: a simple and rapid method to determine the mean cellulose fibril orientation in plants.</a> Journal of microscopy. 198, 101-107 (2000).</li><li>Anderson, C. T., Carroll, A., Akhmetova, L., 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=Real-time+imaging+of+cellulose+reorientation+during+cell+wall+expansion+in+Arabidopsis+roots.">Real-time imaging of cellulose reorientation during cell wall expansion in Arabidopsis roots.</a> Plant physiology. 152, 787-796 (2010).</li><li>Liljegren, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phloroglucinol+Stain+for+Lignin.">Phloroglucinol Stain for Lignin.</a> Cold Spring Harbor Protocols. , (2010).</li><li>Nakano, J., Meshitsuka, G., lin, S., Dence, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Detection of Lignin Methods in Lignin Chemistry. , (1992).</li><li>Sibout, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CINNAMYL+ALCOHOL+DEHYDROGENASE-C+and+-D+are+the+primary+genes+involved+in+lignin+biosynthesis+in+the+floral+stem+of+Arabidopsis.">CINNAMYL ALCOHOL DEHYDROGENASE-C and -D are the primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis.</a> The Plant cell. 17, 2059-2076 (2005).</li><li>Teather, R. M., Wood, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Congo+red-polysaccharide+interactions+in+enumeration+and+characterization+of+cellulolytic+bacteria+from+the+bovine+rumen.">Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen.</a> Applied and environmental microbiology. 43, 777-780 (1982).</li><li>Yeung, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+beginner's+guide+to+the+study+of+plant+structure.">A beginner's guide to the study of plant structure.</a> Proceedings of the 19th Workshop/Conference of the Association for Biology Laboratory Education (ABLE. 19, 365-36 (1998).</li><li>Turner, S. R., Somerville, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collapsed+xylem+phenotype+of+Arabidopsis+identifies+mutants+deficient+in+cellulose+deposition+in+the+secondary+cell+wall).">Collapsed xylem phenotype of Arabidopsis identifies mutants deficient in cellulose deposition in the secondary cell wall).</a> The Plant Cell Online. 9, 689-701 (1997).</li><li>Iiyama, K., Pant, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+the+M&#228;ule+colour+reaction.+Introduction+of+methylated+syringyl+nuclei+into+softwood+lignin.">The mechanism of the M&#228;ule colour reaction. Introduction of methylated syringyl nuclei into softwood lignin.</a> Wood Sci. Technol. 22, 167-175 (1988).</li><li>Yang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+secondary+cell+wall+deposition+in+plants.">Engineering secondary cell wall deposition in plants.</a> Plant biotechnology journal. 11, 325-335 (2013).</li><li>Zhong, R., Ripperger, A., Ye, Z. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectopic+deposition+of+lignin+in+the+pith+of+stems+of+two+Arabidopsis+mutants.">Ectopic deposition of lignin in the pith of stems of two Arabidopsis mutants.</a> Plant physiology. 123, 59-70 (2000).</li><li>Chapple, C. C., Vogt, T., Ellis, B. E., Somerville, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Arabidopsis+mutant+defective+in+the+general+phenylpropanoid+pathway.">An Arabidopsis mutant defective in the general phenylpropanoid pathway.</a> The Plant cell. 4, 1413-1424 (1992).</li><li>Fagard, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PROCUSTE1+Encodes+a+Cellulose+Synthase+Required+for+Normal+Cell+Elongation+Specifically+in+Roots+and+Dark-Grown+Hypocotyls+of+Arabidopsis.">PROCUSTE1 Encodes a Cellulose Synthase Required for Normal Cell Elongation Specifically in Roots and Dark-Grown Hypocotyls of Arabidopsis.</a> The Plant Cell Online. 12, 2409-2423 (2000).</li></ol>

The authors have no financial conflicts to disclose.

Sezioni di gambo A. thaliana sono ampiamente utilizzati per studiare l&#39;organizzazione delle cellule nella parete cellulare secondaria e di esaminare qualitativamente le differenze tra wild type e piante transgeniche. Le tecniche comunemente utilizzate per sezionare i campioni sono di taglio diretto a mano; o quando i campioni sono incorporati in agarosio o fissativo, sezionamento può essere fatto con un vibratomo o microtomo. In contrasto con il taglio manuale, gli ultimi due permettono riducendo il rischi...

La parete cellulare vegetale in possesso di una pletora di informazioni nelle sue varie componenti: la lignina, cellulosa, emicellulosa (silano, glucuronoxylan, xyloglucan, arabinoxilano collegamento misto glucano, o glucomannano), e pectina. Tecniche istologiche forniscono importanti indicazioni visive a studiare le differenze all&#39;interno delle pareti cellulari secondarie a livello organizzativo e cellulari. Varie tecniche istologiche sono stati sviluppati e sono disponibili in letteratura, ma queste tecniche può essere difficile e per principianti richiede tempo perché i protocolli molto dettagliate con semplici istruzioni visive sono quasi mai disponibili. L&#39;obiettivo di questo studio è quello di fornire semplici linee guida per tecniche di colorazione istologiche di ottenere immagini di alta qualità. Sezionare il tessuto stelo è il primo passo nella visualizzazione delle pareti cellulari e forme cellulari. Anche se le sezioni tagliate a mano sono poco costosi e richiedono meno tempo per preparare, l&#39;uso di un vibratome offre coerenza eproduce immagini di alta qualità. Utilizzando una vibratome permette di produrre una migliore qualità dei dati generando anche sezioni con lo stesso spessore, che aiuta a produrre immagini nitide e riduce significativamente il rischio di produrre differenze inesatti tra campioni che deriverebbe semplicemente da una cattiva preparazione del campione. Utilizzando resine di fissare campioni freschi può essere una sfida per principianti e può essere ancora molto tempo, anche per gli esperti quando l&#39;analisi deve essere fatto in fretta. Inoltre, diventa impossibile misurare qualsiasi attività biologica con il campione quando è stato incorporato in una resina. Una semplice tecnica che impiega uno stampo agarosio e fatta in casa è utile per l&#39;incorporamento tessuti dello stelo, e può anche essere utilizzato in altre applicazioni che richiedono dissezione dei tessuti molli. Rispetto al incorporamento esemplari in resina, questo metodo ha il vantaggio di mantenere i tessuti vivi e riducendo la manipolazione dei campioni. Sezionamento dei tessuti attraverso un vibratome è molto preciso e genera homogensezioni unità organizzative, che, a seconda della porta dello studio, possono poi essere utilizzati con diverse tecniche di colorazione diverse. I metodi più semplici per visualizzare lignina e altri aromatici impiegano raggi ultravioletti (UV). Eccitazione delle molecole aromatiche basate sulla luce UV è una vecchia tecnica, ma è ancora uno dei metodi più veloci per la visualizzazione di lignina. Tuttavia, la visualizzazione UV in realtà non è l&#39;ideale per il rilevamento della lignina, perché la luce UV ecciterà altri aromatici. La lignina è costituito principalmente da tre blocchi, i monolignols (alcoli hydroxycinnamyl: alcool coniferilico, alcol sinapyl, e alcool p-coumaryl) 1-3. Macchia Phloroglucinol può fornire indizi per la misura di cinnamaldehydes presenti nello xilema, fibre e tessuti tracheali 4. Phloroglucinol è un buon indicatore di cinnamaldehydes generali e può distinguere tra cinnamaldehydes e altri composti aromatici. I monomeri alcol sinapyl possono essere rilevatie differenziato dall&#39;uso di macchia Maule. Blu di toluidina O è un colorante policromatico e quindi ha la capacità di macchiare diversi elementi della parete cellulare in diversi colori 5,6. L&#39;uso primario di blu di toluidina O è rilevare pectina e lignina 5,6. Il vantaggio di usare blu di toluidina O è che molti elementi della parete cellulare possono essere visualizzati in un unico passaggio. Sia calcofluor bianco e congo rosso sono facili da lavorare e può essere utilizzato per visualizzare cellulosa. Macchie bianche calcofluor cellulosa, callosio, e altri β-glucani non sostituiti o debolmente sostituiti 6-9, mentre congo macchie rosse direttamente al β-(1 → 4)-glucani e in particolare alla cellulosa 10,11. L&#39;obiettivo di questo studio è fornire semplici linee guida per l&#39;utilizzo delle tecniche di colorazione di cui sopra per ottenere immagini di alta qualità da A. thaliana steli.

<table><tbody><tr><td>Agarose</td><td>EMD</td><td>MERC2125</td><td>CAS Number: 9012-36-6</td></tr><tr><td>Phloroglucinol</td><td>Sigma</td><td>P 3502</td><td>1,3,5-trihydroxybenzene [CAS Number: 108-73-6]</td></tr><tr><td>Hydrochloric acid</td><td>EMD</td><td>HX0603-75</td><td>CAS Number: 7647-01-0</td></tr><tr><td>Ammonium hydroxide</td><td>EMD</td><td>AX1303-6</td><td>CAS Number: 1336-21-6</td></tr><tr><td>Toulidine Blue O</td><td>Sigma</td><td>T3260</td><td>Blutene chloride, Tolonium Chloride [CAS Number 92-31-9]&amp;nbsp;</td></tr><tr><td>Potassium permanganate</td><td>Sigma</td><td>223468</td><td>CAS Number 7722-64-7&amp;nbsp;</td></tr><tr><td>Ethanol 190 proof</td><td>KOPTEC</td><td>V1401</td><td>CAS Number: 64-17-5</td></tr><tr><td>Congo Red</td><td>Sigma&amp;nbsp;</td><td>C6277</td><td>Disodium 3,3&#039;-[[1,1&#039;-biphenyl]-4,4&#039;-diylbis(azo)]bis(4-aminonaphthalene 1-sulphonate) [CAS Number 573-58-0]</td></tr><tr><td>Fluorescent Brightener 28/ Calcofluor White Stain</td><td>Sigma</td><td>F3543&amp;nbsp;</td><td>4,4&#039;-Bis[[4-[bis(2-hydroxyethyl)amino]-6-anilino-1,3,5-triazin-2-yl]amino]stilbene-2,2&#039;-disulphonic acid [CAS Number 4404-43-7]&amp;nbsp;</td></tr><tr><td>Vibratome</td><td>Leica</td><td>Leica Vibrating blade microtome VT1000S</td><td>http://www.leicabiosystems.com/products/sectioning/vibrating-blade-microtomes/details/product/leica-vt1000-s/</td></tr><tr><td>Razor</td><td>American Safety razor company</td><td>Item # 60-0139-0000&amp;nbsp;</td><td>Stainless Steel Double Edge Blade (Personna Super)</td></tr><tr><td>Screw Cap Microcentrifuge Tubes (2 ml)</td><td>VWR</td><td>16466-044</td><td></td></tr><tr><td>Microcentrifuge Tubes (0.6 ml)</td><td>Axygen Scientific</td><td>MCT-060-C</td><td></td></tr><tr><td>Mitt</td><td>Bel-Art</td><td>380000000</td><td>SCIENCEWARE&amp;nbsp; Hot Hand Protector Mitt</td></tr><tr><td>Tissue adhesive</td><td>Ted Pella Inc</td><td>10033</td><td>Store at 4 &amp;deg;C or 20 &amp;deg;C for 3 months or longer storage</td></tr><tr><td>Microwave</td><td>Panasonic</td><td>NN-SD762S</td><td>PELCO Pro CA 44 Instant tissue adhesive&amp;nbsp;</td></tr><tr><td>Camera with CCD chip with no mechanical shutter&amp;nbsp;</td><td>Hamamatsu</td><td>C4742-95</td><td></td></tr><tr><td>High speed color camera&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>QImaging</td><td>MicroPublisher 5.0 RTV</td><td></td></tr><tr><td>Camera software&amp;nbsp;</td><td>&amp;nbsp;Molecular Devices</td><td>MetaMorph version 7.7.0.0</td><td></td></tr><tr><td>Imagining anaylsis&amp;nbsp;</td><td>Adobe&amp;nbsp;</td><td>Photoshop CS4</td><td></td></tr><tr><td>Micro Cover Glasses, Square, No. 1</td><td>VWR</td><td>48366-067</td><td>22 x 22 mm (7/8 x 7/8&quot;)-Cover glasses are corrosion-resistant and uniformly thick and flat. No. 1 thickness is 0.13 to 0.17 mm.</td></tr><tr><td>Frosted Micro Slides, 1 mm</td><td>VWR</td><td>48312-003</td><td>75 x 25 mm - 1 mm</td></tr><tr><td>Parafilm M</td><td>Alcan packaging</td><td>BRNDPM998</td><td></td></tr><tr><td>TX2 Filter cube</td><td>Leica</td><td>11513851/11513885</td><td>Filter used for Congo red analysis with a band-pass of 560/40.</td></tr></tbody></table>

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.

Stem Embedding e Sezioni: L&#39;uso dello stampo plastica casalingo per incorporare gli steli in 7% agarosio rivelata facile e veloce (Figura 1). Le due parti (A e B; la figura 1) del sistema flaconcino incorporato rendono semplice per rilasciare facilmente stelo incorporato in agarosio come l&#39;agarosio non si attacca alle parti fiala che sono inerti, mantenendo il sistema pulito. Le fiale possono essere riutilizzati più volte per anni. La comodità di memorizzare stelo incor...

Watch this Scientific Journal Video about Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements at JoVE.com

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.

Colorazione istochimica di<em&gt; Arabidopsis thaliana</em&gt; Cellula secondaria Elementi da parete

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

histochemical-staining-arabidopsis-thaliana-secondary-cell-wall

Research

JoVE Journal

Biology

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

40.4K Views.  Joint Bioenergy Institute. 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.

Video: Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Shootward Movement of CFDA Tracer Loaded in the Bottom Sink Tissues of Arabidopsis

The symplastic tracer 5(6)-carboxyfluorescein diacetate (CFDA) has been widely applied in living plants to demonstrate the intercellular connection, phloem transport and vascular patterning. This protocol shows bottom-to-top carboxyfluorescein (CF) movement in the Arabidopsis by using the root-cutting and the hypocotyl-pinching procedure respectively. These two different procedures result in different efficiencies of CF movement: about 91% appearance of CF in the shoots with the hypocotyl-pinching procedure, whereas only about 70% appearance of CF with the root-cutting procedure. The simple change of loading sites, resulting in significant changes in the mobile efficiency of this symplastic dye, suggests CF movement might be subject to the symplastic regulation, most probably by the root-hypocotyl junction.

Shootward Movement of CFDA Tracer Loaded in the Bottom Sink Tissues of Arabidopsis

The goal of this protocol is to show how to load the CFDA into different sites of the bottom parts of Arabidopsis. We then present the resulting distribution pattern of CF in the shoots.

Movimento shootward di CFDA Tracer caricato nei tessuti lavello inferiore di Arabidopsis

The symplastic tracer 5(6)-carboxyfluorescein diacetate (CFDA) has been widely applied in living plants to demonstrate the intercellular connection, ...

Ricerca

Ambiente

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.

Visualizzazione di lignificazione Dynamics in piante con chimica clicca: doppia etichettatura è beatitudine!

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

Chimica

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, especially, in plant reproductive biology. However, plant morphological, anatomical, and ultrastructural analyses traditionally involve time-consuming embedding and sectioning procedures for bright field, scanning, and electron microscopy. Recent progress in confocal fluorescence microscopy, state-of-the-art 3-D computer-aided microscopic analyses, and the continuous refinement of molecular techniques to be used on minimally processed whole-mount specimens, has led to an increased demand for developing efficient and minimal sample processing techniques. In this protocol, we describe techniques for properly dissecting Arabidopsis flowers and siliques, basic clearing techniques, and some staining procedures for whole-mount observations of reproductive structures.

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

In this protocol, we describe techniques for the proper dissection of Arabidopsis flowers and siliques, some basic clearing techniques, and selected staining procedures for whole-mount observations of reproductive structures.

Intero-monta schiarimento e colorazione delle silique e organi del fiore di Arabidopsis

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, ...

Biologia dello sviluppo

Fluorescent Immunolocalization of Arabinogalactan Proteins and Pectins in the Cell Wall of Plant Tissues

Plant development involves constant adjustments of the cell wall composition and structure in response to both internal and external stimuli. Cell walls are composed of cellulose and non-cellulosic polysaccharides together with proteins, phenolic compounds and water. 90% of the cell wall is composed of&#160;polysaccharides (e.g., pectins) and arabinogalactan proteins (AGPs). The fluorescent immunolocalization of specific glycan epitopes in plant histological sections remains a key tool to uncover remodeling of wall polysaccharide networks, structure and components.
Here, we report an optimized fluorescent immunolocalization procedure to detect glycan epitopes from AGPs and pectins in plant tissues. Paraformaldehyde/glutaraldehyde fixation was used along with LR-White embedding of the plant samples, allowing for a better preservation of the tissue structure and composition. Thin sections of the embedded samples obtained with an ultra-microtome were used for immunolocalization with specific antibodies. This technique offers great resolution, high specificity, and the chance to detect multiple glycan epitopes in the same sample. This technique allows subcellular localization of glycans and detects their level of accumulation in the cell wall. It also permits the determination of spatio-temporal patterns of AGP and pectin distribution during developmental processes. The use of this tool may ultimately guide research directions and link glycans to specific functions in plants. Furthermore, the information obtained can complement biochemical and gene expression studies.

This protocol describes in detail how the plant material for immunolocalization of Arabinogalactan proteins and pectins is fixed, embedded in a hydrophilic acrylic resin, sectioned and mounted on glass slides. We show cell wall related epitopes will be detected with specific antibodies.

Immunolocalizzazione fluorescente delle proteine arabinogalactane e delle pectine nella parete cellulare dei tessuti vegetali

Plant development involves constant adjustments of the cell wall composition and structure in response to both internal and external stimuli. Cell walls ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

Un metodo efficiente per quantitativa, analisi singola cella della cromatina Cambiamento e architettura nucleare in tutto il montaggio Ovuli in<em&gt; Arabidopsis</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Biologia

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.

Risoluzione Quantificazione alta del cristallino cellulosa accumulo in<em&gt; Arabidopsis</em&gt; Roots a Monitor tessuto-specifica delle cellule parete Modifiche

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 ...

Wiesner Staining for Visualizing Lignin: A Technique to Stain Lignin Deposits Present in Stems of Arabidopsis Thaliana

Source: Pradhan Mitra, P. et al., <a href="https://www.jove.com/v/51381">Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements.</a> J. Vis. Exp. (2014).
In this video, we describe the Wiesner staining technique for staining the lignin present in specialized plant cells. This staining technique helps to visualize the lignified cell walls in Arabidopsis thaliana stems and helps to quantify the lignification in the cell walls of different types of cells.

Colorazione Wiesner per la visualizzazione della lignina: una tecnica per colorare i depositi di lignina presenti negli steli di Arabidopsis thaliana

Source: Pradhan Mitra, P. et al., Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements. J. Vis. Exp. (2014).
In this video, we ...

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Biomolecule staining

Maule Staining for Lignin: A Technique to Characterize Lignin Distribution in Arabidopsis Thaliana Stem Sections

Source: Pradhan Mitra, P. et al., <a href="https://www.jove.com/v/51381">Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements.</a> J. Vis. Exp. (2014).
In this video, we describe the Maule staining technique for staining syringyl lignin present in the specialized plant cells. This staining technique helps to characterize the lignin distribution in the xylem and interfascicular fibers.

Colorazione del maule per la lignina: una tecnica per caratterizzare la distribuzione della lignina nelle sezioni dello stelo di Arabidopsis thaliana

Congo Red Staining: A Technique to Visualize Cellulose Deposits in Plant Stem Sections

Source: Pradhan Mitra, P., Loqu&#233;, D. <a href="https://www.jove.com/v/51381">Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements.</a> J. Vis. Exp. (2014).
In this video, we demonstrate the Congo red staining method to visualize cellulose deposits in Arabidopsis thaliana stem sections.

Colorazione con rosso Congo: una tecnica per visualizzare i depositi di cellulosa nelle sezioni del fusto delle piante

Source: Pradhan Mitra, P., Loqu&#233;, D. Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements. J. Vis. Exp. (2014).
In this video, ...

Calcofluor White Staining: A Simple Method for UV-Induced Fluorescence Visualization of Cellulose Deposits in Cell Walls of Dicot Plant Stem Sections

Source: Pradhan M. et al. <a href="https://www.jove.com/v/51381">Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements.</a> J. Vis. Exp. (2014)
In this video, we describe a staining procedure to visualize cellulose deposition within cell walls of Arabidopsis thaliana, a dicot plant, using calcofluor white, which specifically binds to &#946;-linked glucans, exhibiting fluorescence under UV light.

Colorazione bianca Calcofluor: un metodo semplice per la visualizzazione della fluorescenza indotta dai raggi UV dei depositi di cellulosa nelle pareti cellulari delle sezioni del fusto della pianta Dicot

Source: Pradhan M. et al. Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements. J. Vis. Exp. (2014)
In this video, we describe a ...

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