Name: stomata tape-peel: an improved method for guard cell sample preparation
Uploaded: 2018-07-15T13:00:00
Description: Watch this Scientific Journal Video about Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation at JoVE.com

We thank Daniel Chen from the Digital Video Production Team of Buchholz High School for assistance in editing the video. This research on stomatal guard cells, proteomics and metabolomics in the Chen lab has been supported by grants from the US National Science Foundation (0818051, 1158000 and 1412547). Wenwen Kong is supported by the China Scholarship Council. Dr. Qiuying Pang is supported by the China Scholarship Council and National Natural Science Foundation of China (31570396).

1. Growing Plants
<ol>
	<li>Germinate the seeds in potting mixture. Grow plants in a growth chamber with a light intensity of 140 &#181;mol photons m&#8722;2 s &#8722;1 and a photoperiod of 8 h light at 22 &#176;C and 16 h dark at 18 &#176;C for 2 weeks.</li>
	<li>Transplant seedlings of similar sizes individually into 4&#34; diameter pots containing the soil and grow for an additional 3 weeks in the same conditions described in step 1.1. 
	NOTE: Water plants with tap water twice a week to ke...

     

<ol>
	<li>Acharya, B., Assmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormone+interactions+in+stomatal+function.">Hormone interactions in stomatal function.</a> Plant Molecular Biology. 69 (4), 451-462 (2009).</li><li>Schroeder, J. I., Allen, G. J., Hugouvieux, V., Kwak, J. M., Waner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guard+cell+signal+transduction.">Guard cell signal transduction.</a> Annual Review of Plant Physiology and Plant Molecular Biology. 52, 627-658 (2001).</li><li>Obulareddy, N., Panchal, S., Melotto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guard+Cell+Purification+and+RNA+Isolation+Suitable+for+High-Throughput+Transcriptional+Analysis+of+Cell-Type+Responses+to+Biotic+Stresses.">Guard Cell Purification and RNA Isolation Suitable for High-Throughput Transcriptional Analysis of Cell-Type Responses to Biotic Stresses.</a> Molecular Plant-Microbe Interactions. 26 (8), 844-849 (2013).</li><li>Pandey, S., Wang, X. Q., Coursol, S. A., Assmann, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+applications+of+Arabidopsis+thaliana+guard+cell+protoplasts.">Preparation and applications of Arabidopsis thaliana guard cell protoplasts.</a> New Phytologist. 153 (3), 517-526 (2002).</li><li>Schroeder, J. I., Raschke, K., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage+Dependence+Of+K++Channels+In+Guard-Cell+Protoplasts.">Voltage Dependence Of K+ Channels In Guard-Cell Protoplasts.</a> Proceedings of the National Academy of Sciences of the United States of America. 84 (12), 4108-4112 (1987).</li><li>Wu, F. H., 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=Tape-Arabidopsis+Sandwich+-+a+simpler+Arabidopsis+protoplast+isolation+method.">Tape-Arabidopsis Sandwich - a simpler Arabidopsis protoplast isolation method.</a> Plant Methods. 5, 10 (2009).</li><li>Svozil, J., Gruissem, W., Baerenfaller, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteasome+targeting+of+proteins+in+Arabidopsis+leaf+mesophyll,+epidermal+and+vascular+tissues.">Proteasome targeting of proteins in Arabidopsis leaf mesophyll, epidermal and vascular tissues.</a> Frontiers in Plant Science. 6, (2015).</li><li>Schneider, C., Rasband, W., Eliceiri, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9 (7), 671-675 (2012).</li><li>Agnew, B., Murray, D., Patton, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+solid-phase+fluorescence-based+protein+assay+for+quantitation+of+protein+electrophoresis+samples+containing+detergents,+chaotropes,+dyes,+and+reducing+agents.">A rapid solid-phase fluorescence-based protein assay for quantitation of protein electrophoresis samples containing detergents, chaotropes, dyes, and reducing agents.</a> Electrophoresis. 25 (15), 2478-2485 (2004).</li><li>He, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laemmli-SDS-PAGE.">Laemmli-SDS-PAGE.</a> Bio-protocol. Bio101 (80), (2011).</li><li>Thompson, A., 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=Tandem+mass+tags:+A+novel+quantification+strategy+for+comparative+analysis+of+complex+protein+mixtures+by+MS/MS+(vol+75,+pg+1895).">Tandem mass tags: A novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS (vol 75, pg 1895).</a> Analytical Chemistry. 78 (12), 4235-4235 (2006).</li><li>Wiese, S., Reidegeld, K. A., Meyer, H. E., Warscheid, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+labeling+by+iTRAQ:+A+new+tool+for+quantitative+mass+spectrometry+in+proteome+research.">Protein labeling by iTRAQ: A new tool for quantitative mass spectrometry in proteome research.</a> Proteomics. 7 (3), 340-350 (2007).</li></ol>

Guard cells are a model system for studying signal transduction mechanisms in plants and it is important to ensure that the preparation of samples used in the study is the most appropriate to answering biological questions. Despite the increasing interest in guard cells within the plant research community, there is no universal method on how to prepare stomatal guard cells that would allow both the stomatal movement and physiology as well as the molecular changes to be studied in one system. The challenge to isolate them...

Stomata play an important role in the overall physiology and fitness of plants. These tiny plant specific structures are formed by two specialized epidermal cells known as guard cells and are found most abundantly on the abaxial surface of leaves. Stomata are necessary for the exchange of gasses between the plant and the atmosphere, as well as the control of water loss through transpiration. Guard cells regulate stomata aperture through the integration of different external (e.g., light, humidity, pathogen contact, carbon dioxide levels) and internal (e.g., endogenous hormones) stimuli1,2. In the last 20 years, this cell type has become a model system for studying cell signaling processes in plants. These cells make up a small fraction of the total cells in a leaf; thus, to investigate their unique cellular, biochemical and molecular properties in a single-cell manner, it is necessary to isolate them from other leaf cell types. In the past, guard cell studies have often involved the preparation of guard cell protoplasts (GCP)3,4,5. This typically requires the use of large amounts of cell-wall degrading enzymes and or mechanical disruption via blending and has proven to be costly and time-consuming as it generally takes many hours to prepare a GCP sample, often times with little yield. More importantly, because guard cells act as complete pairs to regulate stomata aperture, the use of GCP can be seen as an artificial system to studying guard cell signaling. 
Here, we have developed a method to prepare stomata in which intact guard cells are enriched and maintain physiological responses to stimuli. This method was inspired by a method that was used to isolate mesophyll cell protoplasts6,7. In our method, stomata are prepared using clear Scotch tape to first separate the majority of mesophyll cells attached to the adaxial layer of the leaf from the abaxial layer containing the pavement and guard cells. This is done by affixing a piece of tape to each side of the leaf and peeling them apart. The cells from the abaxial layer are allowed to recover under light in a stomata-opening buffer. Later the remaining pavement cells are removed using a small quantity of cell wall degrading enzymes, leaving behind guard cell that are enriched on the tape. 
In this simple method, stomatal guard cells that are both viable and responsive can quickly and efficiently be prepared, taking less than an hour to obtain enriched stomatal guard cells from 50 peels with minimal cost. The method here imposes less damage to the plant cells than previous methods where protoplasts are prepared. In addition, materials collected from this method yield desirable protein amounts. Most importantly, because the leaf is not subjected to blending or lengthy digestion times and guard cells remain intact, the results of studies will more closely be relevant to that of a guard cell's natural biology. With this method, stomatal movements can be correlated in real time with changes at the molecular level. Thus, the knowledge gained from studies using this preparation method would be important for a more comprehensive understanding of guard cell signaling. 
We used the plant Arabidopsis thaliana as a model; however this protocol can be applied to the enrichment of stomatal guard cells in other plant species such as Brassica napus with small modifications in digestion time. As a proof of concept, we have demonstrated that stomatal guard cells enriched via this method are viable and responsive to stimuli as shown by the fluorescein diacetate (FDA) assay and studies utilizing the plant hormone abscisic acid (ABA), respectively. In addition, we have demonstrated that high-quality proteins can be isolated from these guard cells. Here, we describe a detailed protocol of this process.

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stomata tape-peel: an improved method for guard cell sample preparation

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is often difficult to isolate and thus studying them often provides a challenge. 
This study establishes a stomatal guard cell enrichment method. The protocol utilizes Scotch tape to isolate guard cells and extract proteins from the cells. This method improves the integrity and yield of guard cell during isolation and provides a reliable sample for the study of cell signaling processes and how they correlate to stomatal movement phenotypes. In this method, the plant leaves were partitioned between two portions of tape and peeled apart to remove the abaxial side. This protocol was applied to Arabidopsis leaf tissue to isolate guard cells. The cells were treated with fluorescein diacetate (FDA) to determine viability and with abscisic acid (ABA) to assess stomata movement in response to ABA. In conclusion, this protocol proves useful for preparing enriched stomatal guard cells and isolating proteins. The quality of the cells and proteins obtained here enable physiological and other biological studies.

A representative image of Arabidopsis&#160;guard cells before and after digestion of the mesophyll and epidermal cells is shown in Figure 1. Guard cell viability before and after the removal of mesophyll and epidermal cells can be observed using FDA to measure enzymatic activity and cell membrane integrity (Figure 1B and 1D). Figure 2 illustrates stomatal movement of Arabidopsis in r...

Watch this Scientific Journal Video about Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation at JoVE.com

Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation

This protocol describes a method of preparing enriched stomatal guard cells that is useful for physiological and other biological studies.

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is ...

The overall goal of this experiment is to prepare high quality enriched stomata guard cells for physiological, biochemical, and molecular analyses, such as transcriptomics, proteomics, and metabolomics. Hello, my name is Sheldon Lawrence. I am a graduate student in Dr.Chen's lab in the Department of Biology at the University of Florida.Today, I am going to show you how to isolate guard cells from plant leaf tissue that are useful material for studying both the physiology and biology of stomata. This method is considered a novel method for isolating guard cells. It specifically allows for the enrichment of intact guard cells and provides reliable samples for the study of stomata cell signaling processes.The application of this method can extend to Brassicaceae plants and other plant species with only small modification to the protocol. The demonstration is important as the main step to enrich the guard cells can prove challenging. To begin, select mature, five week old Arabidopsis plants.Carefully remove a leaf with a scalpel, cutting the leaf at the base. Take two pieces of scotch tape and place the excised leaf between them. Gently apply pressure to both sides while ensuring that the end of the tape pieces don't adhere.This can be done by inserting a finger between them. Gently remove the two pieces of tape away from each other, and immediately float the peel taken from the abaxial side of the leaf into stomata opening buffer. After all the peels have been collected, place them under light.Leave them there for two hours. Take several peels and place them on microscope slides. Using a light microscope, adjust the lenses to view the guard cells and ensure that they are open.Place the peels into 50 mls of the cell wall digestion enzyme solution. Float and incubate them at 26 degrees Celsius. After 20 minutes, remove the peels from the enzyme solution and rinse twice briefly with deionized water, and place in fresh stomata opening buffer.Grind the peels in a mortar with liquid nitrogen, with a pestle. Then perform phenol extraction as described in the text. After grinding the peels in a mortar with a pestle, transfer the extract, along with the peels to an Oak Ridge centrifuge tube, and agitate on a shaker for an hour at four degrees Celsius.Precipitate the phenol extracted proteins by adding five volumes of ice-cold 0.1 mol ammonium acetate in 100%methanol. After precipitating the protein overnight at 20 degrees Celsius, harvest the protein by centrifugation and perform pellet washes as described in the text. Re-suspend the pellet in one milliliter of cold acetone, and transfer to a two-milliliter micro centrifuge tube.After centrifugation, discard the acetone supernatant and dry the pellet for ten minutes. Dissolve the protein sample in a dissolution buffer. After vortex and centrifugation, collect the supernatant and measure the protein concentration.Run a SDS-PAGE gel. Visualize the separate protein bands and observe relative protein quality. This is a representative image of Arabidopsis guard cells before and after enzyme digestion of the mesophyll and epidermal cells.Guard cell viability before and after the removal of mesophyll and epidermal cells can be observed using fluorescein diacetate, which measures enzymatic activity and cell membrane integrity. As you can see, guard cells are still viable after peeling and enzyme digestion. This example illustrates stomata movement of Arabidopsis in response to ABA treatment.Guard cells are responsive to treatment and stomata aperture decreases more than 50%after 30 minutes of treatment with ABA. Here is a time course of stomata movement. In addition, a representative image of the stomata aperture at each time point is shown.Protein detection, separation, and relative abundance is observed by SDS-PAGE. Bands are clear and distinct. Four biological replicates are included to emphasize the reproducibility of protein extraction from this method.We first had the idea for this method when we tried to correlate guard cell molecular changes in real time with stomata movement. Once mastered, a protein sample from stomata guard cells can be prepared within a few hours and can be used in multiple experiments. It is important to remember not to inflict a large amount of damage to the guard cells while peeling and handling.Following this method, other techniques can be applied to investigate guard cell signaling, such as isobaric labeling to study redux response of proteins in plants in the normal and stress conditions. After watching this video, you should have a good understanding of how to isolate and enrich intact and viable guard cells, as well as extract high-quality protein appropriate for downstream studies.

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