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 keep the soil moist.</li>
</ol>
2. Preparation of Enriched Stomata
<ol>
	<li>Remove the leaves of 5-week old A. thaliana plants individual with a scalpel.</li>
	<li>Attach each leaf to two pieces of clear Scotch tape, with one piece adhering to the abaxial (lower) side and the other piece adhering to adaxial (upper) side of the leaf. Leave the tape on the leaf for 5 s.</li>
	<li>Gentle peel apart the two pieces of tapes using the index finger and thumb on each hand to separate the abaxial side with pavement and guard cells and the adaxial side with mesophyll cells.</li>
	<li>Place the peels from the abaxial side of the leaves in 60 mL of stomata opening buffer (50 mM KCl, 10 mM MES-KOH, adjusted to pH 6.2 with 1 M KOH) in 40 mm &#215; 12 mm size Petri dishes as they are collected.
	<ol>
		<li>Repeat the above steps until all sample peels are collected.</li>
	</ol>
	</li>
	<li>Place the Petri dishes containing the peels into the growth chamber under light conditions stated in step 1.1. Leave the peels under the light for 2 h to fully open stomata.</li>
	<li>Place peels into a 150 mm x 20 mm Petri dish containing 50 mL of cell wall digesting enzyme mixture (0.7% cellulase R-10, 0.025% macerozyme R-10 , 0.1% (w/v) polyvinylpyrrolidone-40, and 0.25% (w/v) bovine serum albumin in 55% basic solution (0.55 M sorbitol, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.5 mM ascorbic acid, 10 &#181;M KH2PO4, 5 mM 4-morpholineethanesulfonic acid (MES) at pH 5.7 adjusted with 1 M KOH).</li>
	<li>Shake the peels on a reciprocal shaker in a Petri dish at 50 rpm for 20 min.</li>
	<li>Remove peels from enzyme solution with tweezers after 20 min. Transfer the peels to a 150 mm x 20 mm size Petri dish containing 50 mL of water.</li>
	<li>Using a transfer pipette, rinse peels for 15 s twice with 10 mL of water to remove any residual enzyme solution.</li>
	<li>Use tweezers to place peels in a 150 mm x 20 mm size Petri dish with 50 mL of fresh stomata opening buffer as used in step 2.4. Incubate for 1 h in light conditions stated in step 1.1 to allow the stomata to recover.</li>
</ol>
3. Abscisic Acid (ABA) Treatment and Stomata Movement Assay
<ol>
	<li>In a 150 mm x 20 mm size Petri dish. Incubate the enriched stomata peels at 30&#176;C for 15 min in 50 mL of the stomatal opening buffer or in 50 mL of the stomatal opening buffer with a final concentration of 10 &#181;M ABA for the appropriate treatment time.</li>
	<li>Place peels on a shaker at 50 rpm for 5, 15 and 30 min. After each time interval remove a peel with tweezers and follow the imaging steps in step 3.3.</li>
	<li>Take images of stomata before ABA treatment and at the various time points after ABA treatment with a light microscope.
	<ol>
		<li>Take a peel and place it on a glass microscope slide.</li>
		<li>Place the slide on the microscope stage and adjust the stage so that the image of the peel can be visualized.</li>
		<li>Adjust the fine and course focus on the microscope to get a clear image of the guard cells.</li>
		<li>Take several images of multiple stomata at 40X magnification.</li>
	</ol>
	</li>
	<li>Measure 60 stomatal apertures using ImageJ8.</li>
	<li>Calculate the standard error and significance at a p-value &#60; 0.05 of the 60 stomatal aperture measurements.</li>
</ol>
4. Protein Extraction and SDS-Page Separation
<ol>
	<li>Grind the peels for 15 s in enough liquid nitrogen to cover the peels using a chilled mortar and pestle.</li>
	<li>Per 50 peels, add 3 mL of Tris saturated phenol (pH 8.8) and 3 mL protein extraction buffer (0.9 M sucrose, 0.1 M Tris-HCl, 0.01 M EDTA, 0.4% 2-Mercaptoethanol, 10 &#181;L of 1 mM protease and phosphatase inhibitor) with a pipette to the mortar and then grind the peels for 5 additional minutes in a fume hood.</li>
	<li>Transfer the extract using a pipette and the peels using metal tweezers to an Oakridge centrifuge tube and agitate on a shaker at 50 rpm for 1 h at 4 &#176;C.</li>
	<li>Remove the peels from the Oakridge centrifuge tube and centrifuge the extract at 5,000 x g for 10 min. Then transfer the top phenolic phase to a new clean micro-centrifuge tube using a pipette.</li>
	<li>Precipitate the phenol extracted proteins by adding 5 volumes of ice-cold 0.1 M ammonium acetate in 100% methanol. Vortex briefly and incubate overnight at -20 &#176;C.</li>
	<li>Centrifuge at 20,000 x g at 4 &#176;C for 20 min, decant the supernatant by slowly pouring it out of the tube and retain the protein pellet in the tube.</li>
	<li>Wash the protein pellet twice with 0.1 M ammonium acetate in methanol, and then twice with 80% acetone and once with 100% cold acetone. 
	NOTE: Washes are done by adding 10 mL of specified reagent to the tube of protein. Agitate on a shaker at 50 rpm for 5 min, followed by centrifugation at 15,000 x g at 4 &#176;C for 10 min. Then pour the reagent out of the tube retaining only the protein pellet.</li>
	<li>Add 1 mL of 100% cold acetone to the pellet and re-suspend by slowly pipetting up and down.</li>
	<li>Transfer protein suspension via pipette to a 2 mL micro-centrifuge tube then centrifuge at 20,000 x g at 4 &#176;C for 5 min.</li>
	<li>Remove the acetone by decanting it from tube and dry the pellet in fume hood for 10 min.</li>
	<li>Dissolve the protein in a micro-centrifuge tube with 200 &#181;L of dissolution buffer (8 M urea, 0.5% SDS, 30 mM Tris-HCl at pH 8.5) and vortex for 30 min. Centrifuge at 20,000 x g at 15 &#176;C for 20 min and collect the supernatant in a new tube.</li>
	<li>Quantify the protein concentration following the protein quantification protocol9.</li>
	<li>Use protein samples for protein separation by gel electrophoresis following the protocol10.</li>
</ol>

<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>

No conflicts of interest declared.

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.

<table><tbody><tr><td>Stainless Steel Surgical Scalpel</td><td>Feather&amp;nbsp;</td><td>&amp;nbsp;NC9999403</td><td></td></tr><tr><td>Scotch Tape (3M)</td><td>ULINE</td><td>S-9781</td><td></td></tr><tr><td>Petri Dish</td><td>Sigma-Aldrich</td><td>BR455701</td><td></td></tr><tr><td>Cellulase (Onozuka R-10)&amp;nbsp;</td><td>Yakult Pharmaceutical Industry Co., Ltd.</td><td>21560003-3</td><td></td></tr><tr><td>Macerozyme R-10</td><td>Yakult Pharmaceutical Industry Co., Ltd.</td><td>21560003-4</td><td></td></tr><tr><td>DM6000B Microscope&amp;nbsp;</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Protease and Phosphatase Inhibitor Cocktails</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>&amp;nbsp;PI78443</td><td></td></tr><tr><td>Oak Ridge High-Speed PPCO Centrifuge Tubes</td><td>Thermo Fisher Scientific</td><td>3119-0030</td><td></td></tr><tr><td>EZQ Protein Quantitation Kit</td><td>Thermo Fisher Scientific</td><td>R33200</td><td></td></tr><tr><td>Fluorescein Diacetate (FDA)</td><td>Thermo Fisher Scientific</td><td>F1303</td><td></td></tr><tr><td>Abscisic Acid</td><td>Sigma-Aldrich</td><td>A4906</td><td></td></tr><tr><td>Metro Mix 500</td><td>BWI Companies&amp;nbsp;</td><td>TX-500</td><td></td></tr><tr><td>Laemmli Sample Buffer</td><td>Bio-Rad</td><td>161-0737</td><td></td></tr><tr><td>Bio-Safe Comassie (G-250)</td><td>Bio-Rad</td><td>161-0786</td><td></td></tr><tr><td>Microcentrifuge Tube (2mL)</td><td>USA Scientific, Inc.</td><td>1620-2700</td><td></td></tr><tr><td>ImageJ software</td><td>National Institute of Health</td><td></td><td></td></tr><tr><td>Tweezers&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F4017-1EA</td><td></td></tr><tr><td>12% Mini-PROTEAN TGX Precast Gel</td><td>Bio-Rad</td><td>456-1043</td><td></td></tr><tr><td>Microscope slides</td><td>Fisherbrand</td><td>12-550-A3</td><td></td></tr></tbody></table>

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 ...

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

Research

JoVE Journal

Biology

14.9K Views.  University of Florida. Dit protocol beschrijft een methode voor te bereiden van verrijkte stomatal guard cellen die nuttig is voor fysiologische en andere biologische studies.

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

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Immunology and Infection

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

Genetics

mRNA Interactome Capture from Plant Protoplasts

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional gene regulation (PTGR) of messenger RNAs (mRNAs). In the past few years, a number of mRNA-bound proteomes from yeast and mammalian cell lines have been successfully isolated through the use of a novel method called &#34;mRNA interactome capture,&#34; which allows for the identification of mRNA-binding proteins (mRBPs) directly from a physiological environment. The method is composed of in vivo ultraviolet (UV) crosslinking, pull-down and purification of messenger ribonucleoprotein complexes (mRNPs) by oligo(dT) beads, and the subsequent identification of the crosslinked proteins by mass spectrometry (MS). Very recently, by applying the same method, several plant mRNA-bound proteomes have been reported simultaneously from different Arabidopsis tissue sources: etiolated seedlings, leaf tissue, leaf mesophyll protoplasts, and cultured root cells. Here, we present the optimized mRNA interactome capture method for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type that serves as a versatile tool for experiments that include various cellular assays. The conditions for optimal protein yield include the amount of starting tissue and the duration of UV irradiation. In the mRNA-bound proteome obtained from a medium-scale experiment (107 cells), RBPs noted to have RNA-binding capacity were found to be overrepresented, and many novel RBPs were identified. The experiment can be scaled up (109 cells), and the optimized method can be applied to other plant cell types and species to broadly isolate, catalog, and compare mRNA-bound proteomes in plants.

Here, we present an interactome capture protocol applied to Arabidopsis thaliana leaf mesophyll protoplasts. This method critically relies on in vivo UV crosslinking and allows for the isolation and identification of plant mRNA-binding proteins from a physiological environment.

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional ...

Biochemistry

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

Stomatal movement mediates plant gas exchange, which is essential for photosynthesis and transpiration. Stomatal opening and closing are accomplished by a significant increase and decrease in guard cell volume, respectively. Because shuttle transport of ions and water occurs between guard cells and larger neighboring epidermal cells during stomatal movement, the spaced distribution of plant stomata is considered an optimal distribution for stomatal movement. Experimental systems for perturbing the spaced pattern of stomata are useful to examine the spacing pattern's significance. Several key genes associated with the spaced stomatal distribution have been identified, and clustered stomata can be experimentally induced by altering these genes. Alternatively, clustered stomata can be also induced by exogenous treatments without genetic modification. In this article, we describe a simple induction system for clustered stomata in Arabidopsis thaliana seedlings by immersion treatment with a sucrose-containing medium solution. Our method is easy and directly applicable to transgenic or mutant lines. Larger chloroplasts are presented as a cell biological hallmark of sucrose-induced clustered guard cells. In addition, a representative confocal microscopic image of cortical microtubules is shown as an example of intracellular observation of clustered guard cells. The radial orientation of cortical microtubules is maintained in clustered guard cells as in spaced guard cells in control conditions.

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

The goal of this protocol is to demonstrate how to induce clustered stomata in cotyledons of Arabidopsis thaliana seedlings by immersion treatment with a sugar-containing medium solution and how to observe intracellular structures such as chloroplasts and microtubules in the clustered guard cells using confocal laser microscopy.

Stomatal movement mediates plant gas exchange, which is essential for photosynthesis and transpiration. Stomatal opening and closing are accomplished by a ...

Environment

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 ...

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It contains sugars, amino acids, proteins, RNA, lipids and other metabolites. While there is a large interest in understanding the composition and function of the phloem, the role of many of these molecules and thus, their importance in plant development and stress response has yet to be determined. One barrier to phloem analysis lies in the fact that the phloem seals itself upon wounding. As a result, the number of plants from which phloem sap can be obtained is limited. One method that allows collection of phloem exudates from several plant species without added equipment is the EDTA-facilitated phloem exudate collection described here. While it is easy to use, it does lead to the wounding of cells and care has to be taken to remove contents of damaged cells. In addition, several controls to prove purity of the exudate are necessary. Because it is an exudation rather than a direct collection of the phloem sap (not possible in many species) only relative quantification of its contents can occur. The advantage of this method over others is that it can be used in many herbaceous or woody plant species (Perilla, Arabidopsis, poplar, etc.) and requires minimal equipment and training. It leads to reasonably large amounts of exudates that can be used for subsequent analysis of proteins, sugars, lipids, RNA, viruses and metabolites. It is simple enough that it can be used in both a research as well as in a teaching laboratory.

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

Knowledge of the composition of the phloem sap as well as the mechanism of its loading and long-distance transport is essential for the understanding of long-distance signaling in plant development and stress/pathogen response and of assimilate transport. This manuscript describes the collection of phloem exudates using the EDTA-facilitated method.

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It ...

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 ...

Automated Stomatal Response Measurement to Bacterial Invasion in Arabidopsis

Direct Observation and Automated Measurement of Stomatal Responses to Pseudomonas syringae pv. tomato DC3000 in Arabidopsis thaliana

Stomata are microscopic pores found in the plant leaf epidermis. Regulation of stomatal aperture is pivotal not only for balancing carbon dioxide uptake for photosynthesis and transpirational water loss but also for restricting bacterial invasion. While plants close stomata upon recognition of microbes, pathogenic bacteria, such as Pseudomonas syringae pv. tomato DC3000 (Pto), reopen the closed stomata to gain access into the leaf interior. In conventional assays for assessing stomatal responses to bacterial invasion, leaf epidermal peels, leaf discs, or detached leaves are floated on bacterial suspension, and then stomata are observed under a microscope followed by manual measurement of stomatal aperture. However, these assays are cumbersome and may not reflect stomatal responses to natural bacterial invasion in a leaf attached to the plant. Recently, a portable imaging device was developed that can observe stomata by pinching a leaf without detaching it from the plant, together with a deep learning-based image analysis pipeline designed to automatically measure stomatal aperture from leaf images captured by the device. Here, building on these technical advances, a new method to assess stomatal responses to bacterial invasion in Arabidopsis thaliana is introduced. This method consists of three simple steps: spray inoculation of Pto mimicking natural infection processes, direct observation of stomata on a leaf of the Pto-inoculated plant using the portable imaging device, and automated measurement of stomatal aperture by the image analysis pipeline. This method was successfully used to demonstrate stomatal closure and reopening during Pto invasion under conditions that closely mimic the natural plant-bacteria interaction.

Author Spotlight: Advancing Stomatal Research with Automated Aperture Measurement

Here, we present a simple method for direct observation and automated measurement of stomatal responses to bacterial invasion in Arabidopsis thaliana. This method leverages a portable stomatal imaging device, together with an image analysis pipeline designed for leaf images captured by the device.

Stomata are microscopic pores found in the plant leaf epidermis. Regulation of stomatal aperture is pivotal not only for balancing carbon dioxide uptake ...

Relating Stomatal Conductance to Leaf Functional Traits

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, morphological leaf traits have the potential to summarize plants strategies in terms of water use efficiency, growth pattern and nutrient use. The leaf economics spectrum (LES) is a recognized framework in functional plant ecology and reflects a gradient of increasing specific leaf area (SLA), leaf nitrogen, phosphorus and cation content, and decreasing leaf dry matter content (LDMC) and carbon nitrogen ratio (CN). The LES describes different strategies ranging from that of short-lived leaves with high photosynthetic capacity per leaf mass to long-lived leaves with low mass-based carbon assimilation rates. However, traits that are not included in the LES might provide additional information on the species&#39; physiology, such as those related to stomatal control. Protocols are presented for a wide range of leaf functional traits, including traits of the LES, but also traits that are independent of the LES. In particular, a new method is introduced that relates the plants&#8217; regulatory behavior in stomatal conductance to vapor pressure deficit. The resulting parameters of stomatal regulation can then be compared to the LES and other plant functional traits. The results show that functional leaf traits of the LES were also valid predictors for the parameters of stomatal regulation. For example, leaf carbon concentration was positively related to the vapor pressure deficit (vpd) at the point of inflection and the maximum of the conductance-vpd curve. However, traits that are not included in the LES added information in explaining parameters of stomatal control: the vpd at the point of inflection of the conductance-vpd curve was lower for species with higher stomatal density and higher stomatal index. Overall, stomata and vein traits were more powerful predictors for explaining stomatal regulation than traits used in the LES.

Unraveling how physiology and morphology are linked allows a deeper understanding of mechanistic functioning of plant leaves. We present both a procedure to derive parameters of stomatal regulation from stomatal conductance measurements and correlations with traditional functional leaf traits.

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, ...

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant productivity and survival. As such, understanding the mechanisms by which stomata develop and pattern has tremendous agronomic value. This paper describes two phenotypic methods using Arabidopsis cotyledons that can be used to characterize the genes controlling stomatal development and patterning. Presented first are procedures for analyzing the stomatal phenotypes using toluidine blue O-stained cotyledons. This method is fast and reliable and does not require the use of epidermal peels, which are widely used for phenotypic analyses but require specialized training. Due to the presence of multiple cysteine residues, the identification and generation of bioactive EPF peptides that have a role in stomatal development have been challenging. Thus, presented second is a procedure used to identify stomatal ligands and monitor their biological activity by bioassays. The main advantage of this method is that it produces reproducible data relatively easily while reducing the amount of peptide solution and the time required to characterize the role of the peptides in controlling stomatal patterning and development. Overall, these well-designed protocols enhance the efficiency of studying the potential stomatal regulators, including cysteine-rich secretory peptides, which require highly complex structures for their activity.

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

This paper describes two phenotyping methods without the use of epidermal peels to characterize the genes controlling stomatal development. The first method demonstrates how to analyze a stomatal phenotype using a toluidine blue O-stained plant epidermis. The second method describes how to identify stomatal ligands and monitor their biological activities.

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant ...

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

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