Name: arabidopsis thaliana polar glycerolipid profiling by thin layer chromatography (tlc) coupled with gas-liquid chromatography (glc)
Uploaded: 2011-03-18T11:00:00
Duration: 13 min 2 s
Description: Watch this Scientific Journal Video about Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC at JoVE.com

This work is supported by a grant from US National Science Foundation to Christoph Benning.

1. Lipid Extraction
<ol>
<li>Begin lipid extraction by harvesting 30 mg 4-week-old Arabidopsis leaves from plants grown on agar solidified medium or soil and transfer them into 1.5 mL polypropylene reaction tubes. Fresh leaves can be flash frozen in liquid nitrogen and stored at -80 &deg;C.</li>
<li>Add 300 &mu;L extraction solvent composed of methanol, chloroform and formic acid (20:10:1, v/v/v) to each sample. Shake vigorously (using a paint shaker or similar) for 5 minutes.</li>
<li>Add 150 &mu;L of 0.2 M phosphoric acid (H3PO4), 1 M potassium chloride (KCl) and vortex briefly.</li>
<li>Centrifuge at 13,000xg at room temperature for 1 minute. Lipids dissolved in the lower chloroform phase will be spotted onto TLC plates.</li>
</ol>
2. Thin Layer Chromatography (TLC)15
<ol>
<li>To prepare TLC plates, submerge a 20cmx20cm silica gel coated TLC plate with loading strip for 30 sec into 0.15 M ammonium sulfate ((NH4)2SO4) solution, After submerging for 30 seconds, dry the plate for at least 2 days in a covered container. During activation the sublimation of ammonium leaves behind sulfuric acid, which protonates phosphatidylglycerol necessary for its separation from other glycerolipids.</li>
<li>On the day of experiment, activate TLC plates by baking in an oven at 120 &deg;C for 2.5 hours.</li>
<li>After cooling down the activated plates to room temperature, use a pencil to draw a straight line (1.5 cm from the edge of the plate) across the plate at the origin of the chromatogram.</li>
<li>In a fume hood, slowly deliver 3 X 20 &mu;L of lipid extract in the lower chloroform phase using a 20 &mu;L pipette with 200 &mu;L yellow plastic tips under a slow stream of N2. For this purpose, a Tygon Tubing is connected to the regulator of the N2 tank. Keep the spot smaller than 1 cm in diameter. Each plate can hold up to 10 samples (when subsequent GLC analysis is planned).</li>
<li>As the lipid spots completely dry in the fume hood, prepare the developing solvent composed of acetone, toluene, water (91 mL: 30 mL: 7.5 mL). If the ambient relative air humidity is high, separation could be affected. In this case water should be reduced to give (91 mL: 30 mL: 7.0 mL) to achieve the desired separation.</li>
<li>Pour 80 mL developing solvent into a sealable TLC developing chamber (L: H: W=27.0: 26.5: 7.0, cm/cm/cm) and place the plate into the tank with the sample end facing down. Seal the tank using the clamp. The solvent will ascend the plate and lipids will be separated. The development time is approximately 50 minutes at room temperature.</li>
<li>When the solvent front has reached 1 cm from the top of the plate, carefully remove the plate from the tank and dry completely in the fume hood for approximately 10 minutes.</li>
<li>Lipids separated by TLC can be either reversibly stained briefly with iodine for quantitative analysis or irreversibly stained with sulfuric acid or &alpha;-naphthol.
<ol>
<li>Sulfuric acid charring: spray the plate with 50% sulfuric acid in water in a glass spray bottle in the fume hood and bake at 120 &deg;C for 15 minutes (Figure 2A).</li>
<li>&alpha;-naphthol staining for glycolipids: spray the plate with 2.4% (w/v) &alpha;-naphthol in 10% (v/v) sulfuric acid, 80% (v/v) ethanol and bake at 120 &deg;C for 3-5 minutes until glycolipid bands are stained pink or purple (Figure 2B). Overtreatment will lead to charring of all lipids due to presence of sulfuric acid in the reagent.</li>
<li>Iodine staining: in a fume hood, place the plate into a closed TLC tank with iodine crystals (in a tray on the bottom leading to saturation of the atmosphere with iodine vapor until lipids are visible). Don't expose the plate to iodine too long as iodine may covalently modify polyunsaturated fatty acids (Figure 2C). Alternatively, to avoid any oxidation of lipids, only standard lanes interspersed with sample lanes should be stained using a glass wool plugged Pasteur pipette with iodine crystals through which N2 is blown over individual standard lanes.</li>
</ol>
</li>
</ol>
3. Fatty Acyl Methylester (FAME) Reaction16
<ol>
<li>Remove silica surrounding identified lipid spots from the TLC plate with a razor blade. Scrape the lipid containing silica and transfer the silica powder using a funnel into a glass tube with a Teflon (PTFE)-lined screw cap.</li>
<li>Add 1 mL 1 N hydrochloric acid (HCl) in anhydrous methanol to each sample by glass pipette.</li>
<li>Add 100 &mu;L 50 &mu;g mL-1 pentadecanoic acid (15:0) using 200 &mu;L pipette to each sample as internal standard using a 200 &mu;L pipette with 200 &mu;L yellow plastic tip. Keep a tube with only pentadecenoic acid in methanolic HCl as a control. Close glass tubes tightly with Teflon-lined caps.</li>
<li>Incubate glass tubes in an 80 &deg;C water bath for 25 minutes. Tubes need to be sealed so that the solvent does not evaporate.</li>
<li>After tubes have cooled down, add 1 mL 0.9% sodium chloride followed by 1 mL hexane and vortex vigorously. Centrifuge samples at 1000xg for 3 minutes.</li>
<li>In the fume hood, remove the hexane/upper layer of the sample with Pasteur pipette and place it into a new 13x100 mm glass tube.</li>
<li>Evaporate hexane under a slow stream of N2 without drying completely.</li>
<li>Dissolve the resulting fatty acyl methylesters s in 60 &mu;L hexane. Transfer samples into autosampler vials and cap tightly. Samples can be stored at 4 &deg;C for short term and -20 &deg;C for a few days.</li>
</ol>
4. Gas-Liquid Chromatography (GLC)10
<ol>
<li>Before beginning GLC, Ensure that the helium, hydrogen and air cylinders are filled.</li>
<li>Sufficient hexane must be added to the solvent reservoir and the waste container must be empty. For fatty acyl methylesters separation, attach a DB-23 column to the machine.</li>
<li>Place vials into the autosampler. Start the Chemstation software for GLC on the system computer.</li>
<li>Set the inlet temperature at 250 &deg;C with helium flow rate at 48.6 mL min-1 and the pressure at 21.93 psi. The split ratio is 30.0: 1.</li>
<li>The oven temperature is set initially at 140 &deg;C for 2 min and raised to 160 &deg;C at a rate of 25 &deg;C min-1. Then set the temperature to increase from 160 &deg;C to 250 &deg;C at a rate of 8 &deg;C min-1 and hold at 250 &deg;C for 4 min followed by a decrease to 140 &deg;C at a rate of 38 &deg;C min-1. One run takes approximately 21 minutes.</li>
<li>The temperature of the flame ionization detector is 270 &deg;C with a hydrogen flow rate of 30.0 mL min-1, air flow rate at 400 mL min-1 and helium flow rate at 30.0 mL min-1.</li>
<li>Enter the number of vials and sample names in the run sequence table. Set the 10 &mu;L injector to inject 2 &mu;L sample per vial.</li>
<li>When the instrument is ready, initiate the run sequence.</li>
</ol>
5. Representative Results: 
Examples of irreversible staining of TLC-separated lipids from 4-week-old Arabidopsis seedlings are shown in Figure 2. The sulfuric acid stained lipids (Figure 2A) are charred and appear as brown spots. &alpha;-naphthol is preferred to stain glycolipids such as MGDG, DGDG, SQDG etc. Glycolipids stained with &alpha;-naphthol carry a pink-purple color while other polar lipids stain yellow (Figure 2B). The iodine staining is reversible and gives lipids a yellowish color that will disappear over a short time as iodine evaporates (Figure 2C). Briefly iodine stained lipids can be subjected to GLC analysis although unstained lipids are preferable to reduce break down of lipids.
If done successfully, distinctive signals representing different Fatty acyl methylester will be observed after GLC (Figure 3). Fatty acyl methylester with shorter carbon chain and fewer double bonds have shorter retention time using the DB-23 column. Fatty acyl methylester profiling is a sensitive tool to identify mutants with altered lipid composition. In Figure 4, the MGDG18:3 fatty acid molar ratio is decreased in the tgd4-1 mutant compared to the wild type18. By dividing the moles of Fatty acyl methylester for one lipid class with the moles of all lipid classes, the molar ratio of each lipid are calculated. For example, to calculate the molar ratio of MGDG:
(MGDG) mol% = &sum; [FAMEs(MGDG)] / &sum; [FAMEs(total)] x100%
The resulting molar ratios of each lipid class from both the wild type and the mutant can be compared. For instance, the tgd4-1 mutant has increased relative amounts of MGDG and PG but decreased amounts of DGDG and PE (Figure 5)18.

<img src="/files/ftp_upload/2518/2518fig1.jpg" alt="Figure 1" /> 
Figure 1. Flow chart of polar lipid analysis using Arabidopsis seedlings. Total lipids are extracted from 4-week-old Arabidopsis seedlings and separated by TLC. The separated lipids can be scraped from TLC plate for transesterification followed by GLC analysis.
<img src="/files/ftp_upload/2518/2518fig2.jpg" alt="Figure 2" /> 
Figure 2. Separation of lipids on TLC plates. Lipid extracts of 35 mg (fresh weight) wild type seedlings are separated by TLC and stained by sulfuric acid (A), &alpha;-naphthol (B) or iodine vapor (C). Three repeats are shown in each staining method. DGDG, digalactosyldiacylglycerol; MGDG, monogalactosyldiacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; SQDG, sulfoquinovosyldiacylglycerol.
<img src="/files/ftp_upload/2518/2518fig3.jpg" alt="Figure 3" /> 
Figure 3. GLC analysis of Fatty Acid Methylesters (FAMEs) derived from MGDG of the wild type. FAMEs are separated on a 30 m capillary column and detected by flame ionization. Pentadecanoic acid (15:0) is used as an internal standard.
<img src="/files/ftp_upload/2518/2518fig4.jpg" alt="Figure 4" /> 
Figure 4. Fatty acid profile of MGDG in the wild type Col2 (white columns) and the tgd4-1 mutant (black columns). Fatty acids are presented as the number of carbons followed by the number of double bonds. Three repeats are averaged and standard deviations are shown.
<img src="/files/ftp_upload/2518/2518fig5.jpg" alt="Figure 5" /> 
Figure 5. Polar lipids composition of the wild type Col2 (white columns) and the tgd4-1 mutant (black columns). Three repeats are averaged and standard deviations are shown by error bar.

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Natl. Acad. Sci. U. S. A. 97, 10649-10649 (2000).</li><li>James, A. T., MARTIN, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas-liquid+partition+chromatography;+the+separation+and+micro-estimation+of+volatile+fatty+acids+from+formic+acid+to+dodecanoic+acid.">Gas-liquid partition chromatography; the separation and micro-estimation of volatile fatty acids from formic acid to dodecanoic acid.</a> Biochem. 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No conflicts of interest declared.

TLC coupled with GLC provides a robust and rapid tool for quantitative analysis of polar lipids in plants. Small changes in lipid composition can be identified; therefore, this method has been used for large scale screening of mutants impaired in polar lipid metabolic pathways1,20. This method is also widely used for monitoring activities of enzymes utilizing polar lipids as substrate.2,6,7
Besides leaves, the lipid composition of other plant tissues such as roots and seeds or subcellular fractions such as chloroplasts and mitochondria can also be determined in the same way. 
The solvent system (acetone, toluene, water) used here is optimized for the separation of glycolipids and phospholipids in plants. However, in tgd1,2,3,4 mutants and isolated chloroplasts, TGDG runs together with PE while tetragalactosyldiacylglycerol runs with PC. In this case a solvent system with chloroform, methanol, acetic acid and water (85: 20: 10: 4, v/v/v/v) is used13. Sometimes two-dimensional TLC using two different solvent systems is performed to further separate glycolipids and phospholipids19. In addition, plant tissues can be directly subjected to the FAME reaction followed by GLC to determine the total fatty acid profile without initial separation on TLC5. Beside the demonstrated TLC-GLC system, another method used for lipid profiling is based on direct electrospray ionization tandem mass spectrometry17. In this method the initial chromatographic separation of lipids in the extract is omitted. However, this method requires expensive equipment and experienced personnel, which makes it less useful for routine analyses in the lab or for mutant screening.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>&alpha;-naphthol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>N1000</td>
<td>&nbsp;</td>
<tr>
<td>Methanolic HCL 3N</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>33050-U</td>
<td>Dilute to 1N by methanol</td>
</tr>
<tr>
<td>Si250-PA TLC plates</td>
<td>&nbsp;</td>
<td>J.T.Baker</td>
<td>7003-04</td>
<td>With pre-absorbent </td>
</tr>
<tr>
<td>TLC chamber</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>Z266000</td>
<td>&nbsp;</td>
<tr>
<td>Screw cap tubes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>53283-800</td>
<td>&nbsp;</td>
<tr>
<td>Scew caps</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>13-425</td>
<td>&nbsp;</td>
<tr>
<td>PTFE disk</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>200 608</td>
<td>&nbsp;</td>
<tr>
<td>GLC system</td>
<td>&nbsp;</td>
<td>Hewlett Packard</td>
<td>HP6890</td>
<td>&nbsp;</td>
<tr>
<td>DB-23 column</td>
<td>&nbsp;</td>
<td>J&amp;W Scientific</td>
<td>122-2332</td>
<td>&nbsp;</td>
<tr>
<td>GLC vials</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>500 132</td>
<td>&nbsp;</td>
<tr>
<td>Caps of GLC vials</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>201 828</td>
<td>&nbsp;</td>
<tr>
<td>Chemstation software</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>G2070AA</td>
<td>&nbsp;</td>		</tbody>
		</table>
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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.

Watch this Scientific Journal Video about Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC at JoVE.com

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.

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 ...

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MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

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::&amp;#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 ...

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 ...

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 ...

Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli (p)ppGpp Followed by Thin Layer Chromatography

The (p)ppGpp nucleotide functions as a global regulator in bacteria in response to a variety of physical and nutritional stress. It has a rapid onset, in seconds, which leads to accumulation of levels that approach or exceed those of GTP pools. Stress reversal occasions a rapid disappearance of (p)ppGpp, often with a half-life of less than a minute. The presence of (p)ppGpp results in alterations of cellular gene expression and metabolism that counter the damaging effects of stress. Gram-negative and Gram-positive bacteria have different response mechanisms, but both depend on (p)ppGpp concentration. In any event, there is a need to simultaneously monitor many radiolabeled bacterial cultures at time intervals that may vary from 10 seconds to hours during critical stress transition periods. This protocol addresses this technical challenge. The method takes advantage of temperature- and shaker-controlled microtiter dish incubators that allow parallel monitoring of growth (absorbance) and rapid sampling of uniformly phosphate-radiolabeled cultures to resolve and quantitate nucleotide pools by thin-layer chromatography on PEI-cellulose. Small amounts of sample are needed for multiple technical and biological replicates of analyses. Complex growth transitions, such as diauxic growth and rapid (p)ppGpp turnover rates can be quantitatively assessed by this method.

Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli pppGpp Followed by Thin Layer Chromatography

The growth of radiolabeled bacterial cultures in microtiter dishes facilitates high throughput sampling that allows multiple technical and biological replicate assays of nucleotide pool abundance, including that of (p)ppGpp. The effects of growth transitions provoked by sources of physiological stress as well as recovery from stress can be monitored.

The (p)ppGpp nucleotide functions as a global regulator in bacteria in response to a variety of physical and nutritional stress. It has a rapid onset, in ...

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 &lt;em&gt;Arabidopsis thaliana&lt;/em&gt;

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 ...

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.