This work was supported by the Cornell University Agricultural Experiment Station (CUAES) NYC-125433 Hatch Grant and Cornell Start-Up Grant awarded to O.K.V.

Part 1. Preparation of solid medium for growing plants.
We typically culture plants on concentrated Murasige and Skoog (MS) medium supplemented with 1% sucrose and 0.7% agar. It contains all necessary ingredients to keep plants healthy.
For preparing 1L of MS, media pH 5.7
<ol>
	<li>Add 2.15 g of MS powder into 800 ml of water in 1.5 -2.0L autoclavable bottle.</li>
	<li>Place a stirring bar into the bottle and dissolve MS powder by stirring medium on a stirring plate.</li>
	<li>While stirring, add, drop by drop, 1N KOH to adjust pH of MS medium to 5.7.</li>
	<li>Add 10 g of sucrose, keep stirring.</li>
	<li>Add 7 g of agar *.</li>
	<li>Adjust the final volume of the prepared medium to 1000 ml and sterilize by autoclaving.*</li>
	<li>Place autoclaved medium on a stirring plate and cool it down while stirring, so that agar will not precipitate at the bottom of the bottle. Cool down medium to ~ 60 &#176;C.*</li>
	<li>Pour 100 mm Petri plates. Keep plates at 4 &#176;C.</li>
</ol>
*Tip 1: Do not attempt to dissolve agar. It will solubilize during autoclaving. 
*Tip 2: Keep the stirring bar in a bottle during autoclaving, you will need it later. 
*Tip 3: If you can keep you hands on the autoclaved bottle for 10 s, it means that the medium has cooled down sufficiently and you can start pouring plates.
Part 2. Plant material and growth conditions.
<ol>
	<li>Sterilize seeds of Arabidopsis thaliana
	<ol>
		<li>Place 100 mg of seeds into eppendorf microcentrifuge tubes and add 1 ml of 70% ethanol. Mix well and incubate for 2 min. Spin seeds down, aspirate the supernatant.</li>
		<li>Add 1 ml of 1.8 % bleach solution* containing 0.1% Tween-20. Mix for 10 min. Spin down seeds and aspirate bleach solution.</li>
		<li>This step has to be done in a laminar flow hood. Add 1 ml of sterile water to seeds, mix well. Spin down seeds, aspirate water. Repeat this step 5 times.</li>
	</ol>
	</li>
	<li>Spread seeds onto the prepared in step 1 plates (100 seeds/plate).</li>
	<li>Keep seeded plates for 24-48 h at 4 &#176;C in the dark for stratification.</li>
	<li>Grow plants for 14 days in the growth chamber with 8 h light/16 h dark photoperiod (at a photosynthetic photon flux density of 250 &#956;mol m-2s-1) at 23 /19 &#176;C light/dark temperature regime and 75% relative humidity.</li>
</ol>
*Tip 1: Bleach solution is made-up by dilution a household Clorox, containing 6% sodium hypochlorite and adding Tween-20 to a final concentration of 0.1%.
Part 3. Isolation of protoplasts from Arabidopsis seedlings.
<ol>
	<li>Slice 2 g of 14-day-old seedlings with a fresh razor blade in 15 ml of filter-sterilized TVL Solution. We chop plant material in sterile disposable Petri dishes.</li>
	<li>Transfer chopped tissues into 200 ml beaker, add 20 ml of filter-sterilized Enzyme Solution, swirl the beaker to let tissues mix with Enzyme Solution, cover with parafilm and aluminum foil.</li>
	<li>Shake plant tissues at 35 rpm in the dark at room temperature for 16-18 h.</li>
	<li>Collect the released protoplasts into 50 ml Falcon tube by sieving through 8 layers of the cheese cloth, pre-wet in W5 Solution.</li>
	<li>Sieve protoplasts from the cheese-cloth one more time by washing the cloth with 15 ml of W5 Solution.</li>
	<li>Carefully overlay protoplasts with 10 ml of W5 Solution, do not disturb the sugar gradient; centrifuge for 7 min at 100 g.</li>
	<li>Collect 10 ml of protoplasts at the interface of Enzyme Solution and W5 Solution (Fig. 1B) and transfer to a new 50 ml Falcon tube.</li>
	<li>Add 15 ml W5 Solution, centrifuge for 5 min at 60 g. Remove the supernatant.</li>
	<li>Wash protoplasts free from Enzyme Solution by resuspending protoplasts in 15ml of W5 Solution, centrifuge for 5min at 60 g</li>
	<li>Remove the supernatant, resuspend pelleted protoplasts in 1-3ml W5 Solution.</li>
	<li>Evaluate protoplast yield by cell counting with a hemocytometer.</li>
</ol>
Part 4. Reagents:
TVL: 0.3 M sorbitol; 50 mM CaCL2 Filter-sterilize and store at -20 &#176;C.
Enzyme Solution: 0.5 M sucrose, 10 mM MES-KOH [pH 5.7], 20 mM CaCl2, 40 mM KCl, 1% Cellulase (Onozuka R-10), 1% Macerozyme (R10). Filter-sterilize and freshly use.
W5 Solution: 0.1% (w/v) glucose, 0.08% (w/v) KCl, 0.9% (w/v) NaCl, 1.84% (w/v) CaCl2, 2 mM MES-KOH pH 5.7. Filter-sterilize and store at room temperature.
Part 5: Representative Results:
Using Arabidopsis culture conditions described in Parts 1 and 2 renders healthy seedlings, suitable for isolating protoplasts (Fig. 1A). Protoplast are purified by sucrose density gradient centrifugation and collected at the interface of W5 and Enzyme solutions (Fig. 1B). Using protoplast isolation procedure described in Part 3 we typically harvest 5-10 106 of intact protoplasts (Fig. 1C) from one gram of fresh seedlings.
<img alt="Figure 1" src="/files/ftp_upload/1149/1149fig1.jpg" /> 
Figure 1. Isolation of protoplasts from Arabidopsis seedlings. A,Tissues from 14-day-old seedlings of Arabidopsis were collected and converted to protoplasts by a modified procedure of (Chen and Halkier, 2000). B, Protoplasts were purified by sucrose density gradient centrifugation and collected at the interface of enzyme solution and W5 uffer (White arrows). C, Bright-field microscopy of protoplasts. Microphotographs were collected using a cooled CCD camera interfaced with the Zeiss Axioscope 2 plus microscope.

<ol>
	<li>Cocking, E. 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+Method+for+the+Isolation+of+Plant+Protoplasts+and+Vacuoles.">A Method for the Isolation of Plant Protoplasts and Vacuoles.</a> Nature. 187, 962-963 (1960).</li><li>Chen, S., Halkier, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+glucosinolate+uptake+by+leaf+protoplasts+of+Brassica+napus.">Characterization of glucosinolate uptake by leaf protoplasts of Brassica napus.</a> J Biol Chem. 275, 22955-22960 (2000).</li><li>Robert, S., Zouhar, J., Carter, C., Raikhel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+intact+vacuoles+from+Arabidopsis+rosette+leaf-derived+protoplasts.">Isolation of intact vacuoles from Arabidopsis rosette leaf-derived protoplasts.</a> Nat. Protocols. 2, 259-262 (2007).</li><li>Yoo, S. D., Cho, Y. H., Sheen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+mesophyll+protoplasts:+a+versatile+cell+system+for+transient+gene+expression+analysis.">Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis.</a> Nat Protoc. 2, 1565-1572 (2007).</li><li>Zhai, Z., Sooksa-nguan, T., Vatamaniuk, O. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+RNAi+as+a+reverse+genetic+approach+for+gene+functional+analysis+in+protoplasts.">Establishing RNAi as a reverse genetic approach for gene functional analysis in protoplasts.</a> Plant Phys. 149, 642-652 (2009).</li></ol>

The provisional patent describing this procedure for its use in functional analyses of plant genes have been filed with Cornell Center For Technology Enterprise and Commercialization (CCTEC), June 2008. Docket No 50341/015001: Vatamaniuk, O.K., and Zhai, Z. Method for rapid functional analyses of plant genes.

To ensure the high yield of intact protoplast it is very important to start-up with healthy plants. Use filter-sterilize solutions for isolating protoplasts. Remember that protoplasts are fragile. Therefore, when you are handling protoplasts, do not mix, pipet or vortex them vigorously since it will brake them. Instead, mix protoplasts by slowly rotating or taping the centrifuge tube. The described procedure yield intact protoplasts that are viable for at least 96 h. Therefore, protoplasts can be used to study a var...

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<td>Murashige and Skoog Basal Salt Mixture (MS)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M5524</td>
<td>&nbsp;</td>
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<td>Agar</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A1296</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Kreckeler Scientific</td>
<td>82-4001</td>
<td>&nbsp;</td>
<tr>
<td>Cellulase (Onozuka R&#x2010;10)</td>
<td>&nbsp;</td>
<td>RPI Corp</td>
<td>C32200</td>
<td>&nbsp;</td>
<tr>
<td>Macerozyme (R10)</td>
<td>&nbsp;</td>
<td>RPI Corp</td>
<td>M22010</td>
<td>&nbsp;</td>
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<td>Cheesecloth wipes</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>06-665-29</td>
<td>&nbsp;</td>
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<td>Lab Rotator</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>1167152Q</td>
<td>&nbsp;</td>
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<td>Allegra X&#x2010;15R Centrifuge</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>392932</td>
<td>&nbsp;</td>
<tr>
<td>Axioskop2 Plus Microscope</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>&nbsp;</td>
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isolation of protoplasts from tissues of 14-day-old seedlings of arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Watch this Scientific Journal Video about Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana at JoVE.com

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

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35.9K Views.  Cornell University. This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

Video: Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

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 ...

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as phosphatidylcholine or phosphatidylethanolamine, form bilayer membranes which act as both boundaries and interfaces for chemical exchange between cells and their surroundings. Unlike animals, plant cells have a special organelle for photosynthesis, the chloroplast. The intricate membrane system of the chloroplast contains unique glycerolipids, namely glycolipids lacking phosphorus: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)4. The roles of these lipids are beyond simply structural. These glycolipids and other glycerolipids were found in the crystal structures of photosystem I and II indicating the involvement of glycerolipids in photosynthesis8,11. During phosphate starvation, DGDG is transferred to extraplastidic membranes to compensate the loss of phospholipids9,12.
Much of our knowledge of the biosynthesis and function of these lipids has been derived from a combination of genetic and biochemical studies with Arabidopsis thaliana14. During these studies, a simple procedure for the analysis of polar lipids has been essential for the screening and analysis of lipid mutants and will be outlined in detail. A leaf lipid extract is first separated by thin layer chromatography (TLC) and glycerolipids are stained reversibly with iodine vapor. The individual lipids are scraped from the TLC plate and converted to fatty acyl methylesters (FAMEs), which are analyzed by gas-liquid chromatography coupled with flame ionization detection (FID-GLC) (Figure 1). This method has been proven to be a reliable tool for mutant screening. For example, the tgd1,2,3,4 endoplasmic reticulum-to-plastid lipid trafficking mutants were discovered based on the accumulation of an abnormal galactoglycerolipid: trigalactosyldiacylglycerol (TGDG) and a decrease in the relative amount of 18:3 (carbons : double bonds) fatty acyl groups in membrane lipids 3,13,18,20. This method is also applicable for determining enzymatic activities of proteins using lipids as substrate6.

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

Composition of polar lipid extracts and the fatty acid composition of individual glycerolipids are determined in a simple and robust lipid profiling experiment. For this purpose, glycerolipids are isolated by thin layer chromatography and subjected to transmethylation of their acyl groups. Fatty acyl methylesters are quantified by gas-liquid chromatography.

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as ...

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 ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

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

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

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

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

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila tissues often contain a heterogeneous mixture of cell types.&#160;To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...

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 ...

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. Therefore, it is important to establish an efficient transient gene expression system in soybean. Here, we report a simple protocol for the preparation of soybean protoplasts and its application for transient functional analyses. We found that young unifoliate leaves from soybean seedlings resulted in large quantities of high quality protoplasts. By optimizing a PEG-calcium-mediated transformation method, we achieved high transformation efficiency using soybean unifoliate protoplasts. This system provides an efficient and versatile model for examination of complex regulatory and signaling mechanisms in live soybean cells and may help to better understand diverse cellular, developmental and physiological processes of legumes.

We developed a simple and efficient protocol for the preparation of large quantities of soybean protoplasts to study complex regulatory and signaling mechanisms in live cells.