Name: histochemical staining of arabidopsis thaliana secondary cell wall elements
Uploaded: 2014-05-13T23:10:00
Description: Watch this Scientific Journal Video about Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements at JoVE.com

We are thankful to Sabin Russell for editing assistance. This work was part of the DOE Joint BioEnergy Institute (http://www.jbei.org) supported by the U. S. Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the U.S. Department of Energy.

1. Stem Embedding
<ol>
	<li>Make a 7% agarose solution in water (7 g of electrophoresis-grade agarose in 100 ml distilled water). Dissolve the agarose by autoclaving for 20 min or by microwaving for 20 min at lowest intensity (e.g., 10% intensity of a 1,250 watt&#160;microwave).</li>
	<li>Prepare a homemade mold for embedding the stems using plastic vials.
	<ol>
		<li>Using a razor blade, cut off the conical bottom of a 2 ml screw-cap microcentrifuge tube (Part A). With a syringe needle, puncture a hole in the...

<ol>
	<li>Boerjan, W., Ralph, J., Baucher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lignin+biosynthesis.">Lignin biosynthesis.</a> Annual review of plant biology. 54, 519-546 (2003).</li><li>Vanholme, R., Demedts, B., Morreel, K., Ralph, J., 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=Lignin+biosynthesis+and+structure.">Lignin biosynthesis and structure.</a> Plant physiology. 153, 895-905 (2010).</li><li>Humphreys, J. M., Chapple, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rewriting+the+lignin+roadmap.">Rewriting the lignin roadmap.</a> Current opinion in plant biology. 5, 224-229 (2002).</li><li>Adler, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lignin+chemistry&#8212;past,+present+and+future.">Lignin chemistry&#8212;past, present and future.</a> Wood Sci. Technol. 11, 169-218 (1977).</li><li>Brien, T. P., Feder, N., 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=Polychromatic+Staining+of+Plant+Cell+Walls+by+Toluidine+Blue.">Polychromatic Staining of Plant Cell Walls by Toluidine Blue.</a> 59, 368-373 (1964).</li><li>Mori, B., Bellani, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+staining+for+cellulosic+and+modified+plant+cell+walls.">Differential staining for cellulosic and modified plant cell walls.</a> Biotechnic &amp; histochemistry: official publication of the Biological Stain Commission. 71, 71-72 (1996).</li><li>Maeda, H., Ishida, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+of+binding+of+hexopyranosyl+polysaccharides+with+fluorescent+brightener.">Specificity of binding of hexopyranosyl polysaccharides with fluorescent brightener.</a> Journal of biochemistry. 62, 276-278 (1967).</li><li>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=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>

A. thaliana stem sections are widely used to study the organization of the cells in the secondary cell wall and to qualitatively examine the differences between wild type and transgenic plants. The commonly used techniques for sectioning the specimens are direct hand cutting; or when specimens are embedded in agarose or fixative, sectioning can be done with a vibratome or microtome. In contrast to hand cutting, the last two allow reducing the risk of producing inaccurate differences between samples that would be...

The plant cell wall holds a plethora of information within its various components: lignin, cellulose, hemicelluloses (xylan, glucuronoxylan, xyloglucan, arabinoxylan, mixed linkage glucan, or glucomannan), and pectin. Histological techniques provide important visual cues in studying differences within the secondary cell walls at organizational and cellular levels. Various histological techniques were developed and can be found in the literature, but these techniques can be challenging and time-consuming for beginners because very detailed protocols with simple visual instructions are seldom if ever available. The goal of this study is to provide simple guidelines for histological staining techniques to obtain high-quality images.
Sectioning the stem tissue is the first step in visualization of cell walls and cell shapes. Although hand-cut sections are inexpensive and take less time to prepare, use of a vibratome offers consistency and yields high-quality images. Using a vibratome allows producing better data quality by generating even sections with the same thickness, which helps to produce sharp images and reduces significantly the risk of producing inaccurate differences between samples that would be simply caused by a bad sample preparation. Using resins to fix fresh specimens can be a challenge for beginners and may still be time-consuming even for experts when an analysis needs to be done quickly. In addition, it becomes impossible to measure any biological activity with the sample when it has been embedded in a resin. One simple technique that employs agarose and a homemade mold is helpful for embedding stem tissues, and it can also be utilized in other applications requiring dissection of soft tissues. Compared to embedding specimens in resin, this method has the advantage of keeping the tissues alive and reducing sample manipulation. Sectioning tissues via a vibratome is highly precise and generates homogenous sections, which, depending on the goal of the study, can then be used with several different staining techniques.
The simplest methods to visualize lignin and other aromatics employ ultraviolet (UV) light. Excitation of aromatic-based molecules by UV light is an old technique, but it is still one of the quickest approaches for visualization of lignin. However, UV visualization actually is not ideal for lignin detection because UV light will excite other aromatics. Lignin is made up primarily of three building blocks, the monolignols (hydroxycinnamyl alcohols: coniferyl alcohol,&#160;sinapyl alcohol,&#160;and p-coumaryl alcohol)1-3. Phloroglucinol stain can provide clues to the extent of cinnamaldehydes present in the xylem, fiber, and tracheal tissues4. Phloroglucinol is a good indicator of general cinnamaldehydes and can differentiate between cinnamaldehydes and other aromatics. The sinapyl alcohol monomers can be detected and differentiated by the use of M&#228;ule stain. Toluidine blue O is a polychromatic dye and therefore has the ability to stain different elements of the cell wall in different colors5,6. The primary use of toluidine blue O is to detect pectin and lignin5,6. The advantage of using toluidine blue O is that many elements of the cell wall can be visualized in a single step. Both calcofluor white and congo red are easy to work with and can be used to visualize cellulose. Calcofluor white stains cellulose, callose, and other non-substituted or weakly substituted &#946;-glucans6-9, whereas congo red stains directly to &#946;-(1&#8594;4)-glucans and particularly to cellulose10,11. The goal of this study is to provide simple guidelines for the use of the aforementioned staining techniques to obtain high-quality images from A. thaliana stems.

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	<tbody>
		<tr>
			<td>&#160;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>
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			<td>Ammonium hydroxide</td>
			<td>EMD</td>
			<td>AX1303-6</td>
			<td>CAS Number: 1336-21-6</td>
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		<tr>
			<td>Toulidine Blue O</td>
			<td>Sigma</td>
			<td>T3260</td>
			<td>Blutene chloride, Tolonium Chloride [CAS Number 92-31-9]&#160;</td>
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		<tr>
			<td>Potassium permanganate</td>
			<td>Sigma</td>
			<td>223468</td>
			<td>CAS Number 7722-64-7&#160;</td>
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			<td>Ethanol 190 proof</td>
			<td>KOPTEC</td>
			<td>V1401</td>
			<td>CAS Number: 64-17-5</td>
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			<td>Congo Red</td>
			<td>Sigma&#160;</td>
			<td>C6277</td>
			<td>Disodium 3,3&#39;-[[1,1&#39;-biphenyl]-4,4&#39;-diylbis(azo)]bis(4-aminonaphthalene 1-sulphonate) [CAS Number 573-58-0 ]</td>
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			<td>Fluorescent Brightener 28/ Calcofluor White Stain</td>
			<td>Sigma</td>
			<td>F3543&#160;</td>
			<td>4,4&#39;-Bis[4-[bis(2-hydroxyethyl)amino]-6-anilino-1,3,5-triazin-2-yl]amino]stilbene-2,2&#39;-disulphonic acid [CAS Number 4404-43-7]&#160;</td>
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			<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>
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			<td>Razor</td>
			<td>American Safety razor company</td>
			<td>Item # 60-0139-0000&#160;</td>
			<td>Stainless Steel Double Edge Blade (Personna Super)</td>
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			<td>Screw Cap Microcentrifuge Tubes (2ml)</td>
			<td>VWR</td>
			<td>16466-044</td>
			<td></td>
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			<td>Microcentrifuge Tubes (0.6ml)</td>
			<td>Axygen Scientific</td>
			<td>MCT-060-C</td>
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			<td>Mitt</td>
			<td>Bel-Art</td>
			<td>380000000</td>
			<td>SCIENCEWARE&#160; Hot Hand Protector Mitt</td>
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			<td>Tissue adhesive&#160;</td>
			<td>Ted Pella Inc</td>
			<td>10033</td>
			<td>Store at 4&#176;C or 20&#176;C for 3 months or longer&#160; storage&#160;</td>
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			<td>Microwave</td>
			<td>Panasonic</td>
			<td>NN-SD762S</td>
			<td>PELCO Pro CA 44 Instant tissue adhesive&#160;</td>
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			<td>Camera with CCD chip with no mechanical shutter&#160;</td>
			<td>Hamamatsu</td>
			<td>C4742-95</td>
			<td></td>
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			<td>High speed color camera&#160;&#160;&#160;</td>
			<td>QImaging</td>
			<td>MicroPublisher 5.0 RTV</td>
			<td></td>
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			<td>Camera software&#160;</td>
			<td>&#160;Molecular Devices</td>
			<td>MetaMorph version 7.7.0.0</td>
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			<td>Imagining anaylsis&#160;</td>
			<td>Adobe&#160;</td>
			<td>Photoshop CS4</td>
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			<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&#34;)-Cover glasses are corrosion-resistant and uniformly thick and flat. No. 1 thickness is 0.13 to 0.17mm.&#160;</td>
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			<td>Frosted Micro Slides, 1mm</td>
			<td>VWR</td>
			<td>48312-003</td>
			<td>75 x 25 mm- 1mm</td>
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			<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>
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			<td>Parafilm M</td>
			<td>Alcan packaging</td>
			<td>BRNDPM998</td>
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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 and Sectioning: 
The use of the homemade plastic mold to embed the stems in 7% agarose proved to be fast and easy (Figure 1). The two parts (A and B; Figure 1) of the embedded vial system make it simple to easily release the stem embedded in agarose as the agarose does not stick to the vial parts that are inert, keeping the system clean. The vials can be reused multiple times for years. The convenience of storing the embedded stem also makes the next steps easier. In ...

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.

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 ...

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

Research

JoVE Journal

Biology

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

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.

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 ...

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light conditions, which are preferably variable in quantity and quality so that studies under different setups can be conducted. These requirements are met by climatic chambers featuring light emitting diodes (LED) lights, which can &#8211; in contrast to fluorescent lights &#8211; be set to different wavelengths. LEDs are energy conserving and emit virtually no heat even at light intensities, which often constitutes a problem with other light sources. The presented protocol provides a step-by-step guidance of how to program a climatic chamber equipped with variable LED lights as well as describing several approaches for in depth analysis of growth phenotypes. Depending on the experimental set-up various characteristics of the growing plants can be observed and analyzed. Here we describe how to determine fresh weight, leaf area, photosynthetic activity, and stomatal density. We demonstrate that in order to obtain reliable data and draw valid conclusions it is mandatory to use a sufficient number of individuals for statistical evaluation. Taking too few plants for this kind of analysis results in high statistical errors and consequently in less clear interpretations of the data.

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Here, we present a protocol for studying plant growth behavior and especially phenotypes in a reproducible manner. We show how to provide variable and at the same time stable light conditions. Proper analyses depend on sufficient sample numbers and valid statistical evaluations.

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light ...

Spatiotemporal Investigation of ERL1 Dynamics via Confocal Imaging

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

Author Spotlight: Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells  

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...

An Improved Method to Measure Pollen Hydration Profiles in Arabidopsis thaliana

A High-Resolution, Single-Grain, In Vivo Pollen Hydration Bioassay for Arabidopsis thaliana

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is established between the interacting partners. Studies across a range of species have revealed that a series of molecular checkpoints regulate the pollen-stigma interaction to ensure that only compatible, generally intraspecific pollen is successful in effecting fertilization. In species that possess a 'dry stigma', such as the model plant Arabidopsis thaliana, the first post-pollination, prezygotic compatibility checkpoint is the establishment of pollen hydration. 
This phase of pollination is tightly regulated, whereby signals from the pollen grain elicit the release of water from the stigma, thus permitting pollen hydration. The ability to accurately measure and track pollen hydration over time is key to the design of experiments directed at understanding the regulation of this critical step in reproduction. Published protocols frequently utilize flowers that have been excised from the parent plant, maintained on liquid or solid media, and bulk pollinated. 
This paper describes a noninvasive, in vivo pollination bioassay that permits minute-by-minute hydration tracking of individual A. thaliana pollen grains at high resolution. The assay is highly reproducible, able to detect very subtle variations of pollen hydration profiles, and thus is suitable for the analysis of mutants that affect pathways regulating pollination. Although the protocol is lengthier than those described for bulk pollinations, the precision and reproducibility it provides, along with its in vivo nature, make it ideal for the detailed dissection of pollination phenotypes.

Author Spotlight: A High-Resolution, Single-Grain, In Vivo Pollen Hydration Bioassay for Arabidopsis thaliana  

An improved method to measure pollen hydration profiles in Arabidopsis thaliana is described here. The new method offers higher resolution, is noninvasive, and is highly reproducible. The protocol represents a new tool for a finer dissection of the processes that regulate the early stages of pollination.

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is ...

Growth of Arabidopsis thaliana Plants and Preparation of Flowers for Pollen Hydration Assay

Growth of Arabidopsis thaliana Plants and Preparation of Flowers for Pollen Hydration Assay   

Begin by stratifying A.thaliana seeds in sterile Milli-Q water for three days at four degrees Celsius. Transfer the stratified seeds to the compost pots by pipetting the stratified seeds. Place the pots in an environmentally controlled growth chamber for approximately six weeks until the inflorescences are well established.To ensure synchronous flowering, sow the pollen donor and recipient plants and any other appropriate control plant lines together. One day prior to the bioassay, select these stage 12 floral buds from the recipient plant, Having unopened flower buds that will show the opening of the flower and the anther dehiscence on the following day. Next, place a glass slide under a stereo dissecting microscope.Tape the region of the stem close to the selected recipient flower on that glass slide. Using a pair of fine-tipped forceps, carefully tease open the flower bud and remove all the flower petals and anthers. Once done, ensure that the pistil is undamaged and the stigma is free of contaminating pollen.

Pollen Hydration Assay in Arabidopsis thaliana: Raw Data Acquisition and its Measurements

Pollen Hydration Assay in Arabidopsis thaliana: Raw Data Acquisition and its Measurements  

Begin the assay by laying the labeled A.thaliana pollen recipient plant on its side, positioning the emasculated flower on the stage of an inverted microscope to image the stigma. To fix the position of the emasculated recipient flower, immobilize the stem using strips of masking tape. From the pollen donor plant, remove a healthy and freshly-opened flower and place the pollen donor plant under a dissecting microscope.Gather pollen grains using fine-tipped forceps. Remove excess pollen grains by lightly touching the forceps against the donor petals until the pollen grain's monolayer is formed. Return to the pollen recipient plant and use a low power objective lens to focus the inverted microscope on the stigma.Holding the forceps along the opening between the arms of the forceps, carefully approach and unpollinated stigmatic papilla cell. Continue approaching the unpollinated stigmatic papilla cell until the suitably positioned pollen grain on the forceps makes light contact with its surface. Once pollen attachment is confirmed, slowly withdraw the forceps.Orient the pollen grain with its equatorial axis visible and in sharp focus, and immediately switch to a higher power objective lens, like 20x. Capture the first pollen grain image as T-0, and continue capturing images at one-minute intervals for 10 minutes. Adjust the focus to accommodate small movements in the pollen grain or stigma.Record the room's ambient temperature and relative humidity every two minutes, allowing future comparisons between experimental replicates. Save the images in a lossless format, such as ND2, before repeating the pollination and imaging steps for additional pollen grains to acquire data for control and experimental pollinations. Begin the data measurements for pollen hydration assay using image analysis software.Use similar parameters of digital zoom and the approach defining the pollen boundary for all measurements in the datasets. Record the semiminor axis values for all time points in the dataset. Once the measurements are complete for a dataset, export the raw semiminor axis values of each individual time point to a spreadsheet.Present the data in columns per image stack and calculate the percentage semiminor axis change. Repeat the steps and obtain data from at least 15 hydrated pollen grains for each plant line. A few pollen grains may fail to hydrate or may hydrate significantly slower than expected.They're known as dud grains. Exclude these from the dataset. Calculate the mean values for each time point per plant using the specialized software package GraphPad Prism.Analyze the hydration data from wild-type and mutant lines at each time point using unpaired T-test and one-way ANOVA to compare the means of the specific time point of interest. To compare the means across all the time points, perform multiple T-tests between wild-type and mutant lines across multiple time points. The pollen hydration time series data for wild-type plants collected on different days showed that the minimum and maximum values for the means between replicates for all time points were within 3%This representative data for wild-type pollinations demonstrated a high degree of consistency for relatively low sample numbers and across different days.

﻿Preparation of Dual-Colored F1 Transgenic Arabidopsis thaliana

Preparation of Dual-Colored F1 Transgenic Arabidopsis thaliana  

To begin, grow homozygous transgenic Arabidopsis thaliana plants bearing ERL1-YFP and a red fluorescent protein, or RFP-labeled marker protein RFP ARA7 side-by-side in a growth room until flowering. On the young inflorescence of plant A, remove all the older flowers and calyx, except for one recently opened flower. Carefully remove the younger flowers and floral merostems.In the recently opened flower, remove the sepals, petals, and stamen using a pair of sharp tweezers, leaving only the pistil on the inflorescence. From plant B, take mature stamen from an opened flower and deposit the pollen grains into the stigma of the dissected pistil of plant A.Label this manually crossed flower, indicating its parents and the date of the genetic cross. When the calyx turns yellow or brown, harvest the F1 seeds to check for a successful F1 plant having yellow fluorescent protein, or YFP, and RFP signals.