This work was supported by the National Science Foundation NSF-IOS grant#1144391 to SHB.

1. Preparation of Plants
<ol>
	<li>Arabidopsis seeds can be germinated directly on soil or on MS plates. Grow the plants for four to six weeks in growth chambers with a 12-hr&#160;photoperiod (22 &#176;C day, 15 &#176;C night14). Water plants once to twice a week. Use bottom tray for watering; do not water plants from the top; allow the trays to dry before re-watering. Make sure plants are well hydrated at time of harvest.</li>
</ol>
2. Preparation of Solutions and Dishes
<ol>
	<li>K2-EDTA solution: Make a 100 mM K2-EDTA solution stock solution, which can be kept in the refrigerator. On the day of the collection, dilute the solution one to five (one part EDTA solution, four parts water) to obtain a 20 mM K2-EDTA solution.</li>
	<li>Fill a glass or Petri dish (diameter 7-15 cm) halfway with the 20 mM K2-EDTA solution. If collecting samples from different treatments (for example control and salt-stressed plants or different genotypes), prepare separate dishes for each treatment.</li>
	<li>Fill desired number of 1.7 ml reaction tubes with 1.4 ml of 20 mM K2-EDTA solution. Fill an equal number of reaction tubes with 1.4 ml autoclaved deionized or Millipore water.</li>
	<li>Put paper towels on the bench to set plants on; get one new razor blade and put on gloves.</li>
</ol>
3. Collection of Phloem Exudates (Depicted in Flowchart in Figure 1)
<ol>
	<li>Harvest rosette leaves from four to six week-old plants by cutting them with the razor blade at the base of the petiole close to the center of the rosette.</li>
	<li>Immediately place the leaves in the dishes containing 20 mM K2-EDTA. Make sure the cut end of the petiole is submerged in the solution (Figure 2).</li>
	<li>Once 15 leaves have been collected (ca. three to four plants), gently stack leaves on top of each other such that the cut petioles are aligned with each other. Recut the base of the petioles (ca. 1 mm) and immediately transfer the leaves into one of the reaction tubes with the 20 mM K2-EDTA solution. The leaves fit easiest if they are slightly rolled up. Avoid squeezing the leaves in since this might cause injury of cells and, thus, leads to the collection of leaf cell content.</li>
	<li>If more material is needed, gently cover the samples with a wet paper towel. Use a new set of dishes if for the collection of samples from different treatments or genotypes.</li>
	<li>Once all the samples are positioned in reaction tubes, collect the exudates either in the dark or in the light.</li>
	<li>For collection in the dark, line the floor of a large shallow dish with wet paper towels. Place the rack with the leaf-filled reaction tubes in the dish and either cover with wet paper towels or put into a black plastic back with slits for aeration. Place the entire setup in a cabinet or a dark room.</li>
	<li>For collection in the light, line the bottom of a clear plexiglass container with wet paper towels. Place the rack with the leaf-filled reaction tubes in the container and close it.</li>
	<li>After 1 hr, remove leaves gently from the container and the reaction tubes and wash thoroughly with distilled, deionized or Millipore water to remove all EDTA. This step serves three purposes: (1) it removes the EDTA to minimized damage to the cells, (2) it eliminates the interference of EDTA with SDS-PAGE and chromatography, and (3) removes any compounds that have accumulated from cut/damaged cells. Immediately transfer into the prepared reaction tubes containing autoclaved water and return to the humidified collection containers for the collection of exudates. At this point, add RNase inhibitor if the exudate needs to be used for RNA extraction. Optional: Proteinase inhibitor can be added if protein analysis is desired.</li>
	<li>After the intended collection time, pull out and discard the leaves and freeze the phloem exudates in liquid N2 for further use. If extended storage is needed, lyophilize the samples and store at -80 &#176;C.</li>
	<li>In the case of Arabidopsis, metabolites can already be detected after collection of exudates for 1 hr. However, collection for 5-8 hr&#160;is advisable if analysis of less abundant components is planned. For larger plants like Perilla, longer collection times (8 hr) are recommended. 
	Exudate collected from 15 leaves is typically enough for one lane on an SDS-PAGE gel (proteins), for mRNA extraction, GC-MS (sugars and metabolites). For lipid analysis the pooling of 2-3 samples (exudate from 30-45 leaves) is recommended and leads to clearer results.</li>
</ol>
4. Subsequent Analysis (For Video We Only Show 4.4)
<ol>
	<li>For protein analysis, resuspend sample in 15 &#956;l water and 30 &#956;l L&#228;mmli buffer, heat to 95 &#176;C for 5 min, quickly spin down any precipitates and load entire sample onto a SDS-PAGE gel (45 &#956;l loading pockets). Samples should be stained with Colloidal Coomassie or other sensitive stains since protein abundance is very low.</li>
	<li>For sugar and small metabolite analysis, add derivatizing agents to the dried sample as described in the literature14.</li>
	<li>mRNA can be analyzed using RT-PCR, microarray or RNA-Seq analysis.</li>
	<li>For lipid analysis immediately pool two to three samples and partition against Chloroform:Methanol (1:1) in the fume hood as follows: add an equal amount of Chloroform: Methanol to each sample, vortex for a few seconds, and centrifuge for 4 min&#160;at 400 x g. Collect the bottom (organic) phase in a glass tube with teflon cap and repeat partition with the top phase three more times. Dry the combined organic phases under a stream of N2 and submit to further analysis (Thin-layer chromatography: TLC14, 37; Liquid chromatography-Mass spectrometry: LC-MS14).</li>
</ol>

We have nothing to disclose.

The EDTA-facilitated collection of phloem exudates is very simple, needs minimal equipment, and is applicable to most plants. Overall, this method extends the ability to analyze phloem exudates from more species. Though the exudate is diluted, it is easy to collect many different samples or from many plants to scale up to a large amount of material. This then allows for the detection of novel compounds that are in very low abundance and would otherwise be overlooked.
This method can be used for multiple plant species and works as described for the ones listed in this manuscript. If new species are explored, the two aspects that might need modification are the number of leaves used and the time of exudation. In most cases, a five to eight hour exudation should be suitable. To confirm the use of this method for a new plant species, exudate needs to be collected and one or more of the subsequent analysis as described in protocol part 4 needs to be performed. Depending on the intensity of the signal observed, the collection volume may need to be scaled up. In general, lipid and protein analysis will require more material than sugar and metabolite analysis since the former are less abundant in phloem exudates.
Because the EDTA-facilitated exudate collection involves cut surfaces as well as a potentially damaging effect of EDTA, several points have to be considered: (1) after the one hour incubation with EDTA, the petioles have to be washed thoroughly. This removes any compounds obtained from wounded cells as well as the EDTA itself, which could damage cells or interfere with the extraction. (2) Positive and negative controls need to be included to ensure data obtained are derived from phloem sap and not from injured cells (see critical steps below). (3) Phloem sap does contain several enzymes, which could be active during exudate collection. Hence, in some cases it may be useful to collect for different amounts of time. (4) Since the method involved exudation into water, an absolute quantification of the compounds is not possible.
Critical steps: The key to the successful use of this method is the use of caution while handling the sample to not injure any cells as well as the use of appropriate controls (see ref. 14). One suitable control is the collection of phloem exudates without prior treatment with EDTA. In this case the phloem will seal itself and no exudate can be collected. Any contaminating compounds coming from the wounded cells will be detected here. A second control would be the analysis of sugars in the exudate. In Arabidopsis, sucrose should be the predominant metabolite within the phloem sap, with fructose to sucrose ratio of between 1:4 and 1:8 (ref 8, 14).
For RNA extraction the use of an RNAse inhibitor is necessary. In addition, a positive control of previously described phloem-localized mRNA (UBC9: Ubiquitin conjugating enzyme 9, At4g27960; 8, 14) and a negative control of for example a chloroplast mRNA (Rubisco LSU) are advisable.
Because the exudate contains active enzymes, a time course is advised to make sure changes in the phloem profile are not simply due to variations in the collection time. In some cases, it may be beneficial to use proteinase inhibitors. However, the investigators need to keep in mind, that the collected exudate will be concentrated and that any added compounds will be largely concentrated. A second critical step within the protocol is the recutting of the petiole under EDTA. This step serves a dual purpose: First, it removes any sealed sieve plates while preventing the formation of new plugs thus allowing for free flow of the phloem sap. Second, it removes the cavitation bubble in the xylem generated while cutting and thus allows for xylem transport. The size of the second cut depends on the plants used. In plants with larger petioles it should be several millimeters (up to 1 cm) above the first cut, in plants like Arabidopsis, 1-2 mm are sufficient.
The use of this method can lead to the discovery of novel compounds in phloem exudates that could have signaling, developmental or biomedical implications. It has prompted a more in-depth look at long-distance lipid signaling, which was a little-studied field within plant science due to the lack of access to the phloem.

Plants cannot move to escape adverse conditions. Consequently, they had to develop mechanisms to detect environmental stresses, elicit and transmit a related signal throughout the plant, and adjust development accordingly. Two transport systems exist for the distribution of water, nutrients and other (signaling) compounds. The first is the xylem; which typically transports water and minerals taken up by the roots throughout the plant. The second is the phloem. The view of the phloem has changed from a simple assimilate transport system to a conduit for RNA, protein, viruses, lipids and other small molecules. It plays an important role in assimilate and nutrient transport, response to biotic and abiotic stress, as well as in plant growth and development. It is now called the &#34;information superhighway&#34; of the plant18.
The phloem is comprised of several cell types: phloem parenchyma, as well as specialized companion cells and sieve elements. The sieve element is the site of long-distance movement. To allow for unobstructed longitudinal flow, sieve elements are missing most organelles as well as nuclei and are thought to contain at best a limited translational machinery21, 33. It is believed that companion cells synthesize proteins and other compounds, which travel in the phloem stream. These compounds are then transported into the sieve element via plasmodesmata and can function as long distance signals 4,16.
Several groups of compounds can be found in phloem exudates:
<ul>
	<li>Sugars are often the products of photosynthesis and are transported as &#34;energy-carrying&#34; molecules to other parts of the plant for storage or as building blocks. However, they can also function as antifreeze or as signaling compounds.</li>
	<li>Proteins can also be found in phloem exudates. They include metabolic enzymes but also have signaling function. One example of a protein which serves as a developmental signal is the Flowering locus T protein, which signals the induction of flowering as well as seasonal leaf abscission in plants.6</li>
	<li>Nucleic acids are present in phloem exudates in the form of mRNA, small and micro RNAs, and viral RNAs. They appear to be largely involved in signaling 27, 28, 39. At the same time, rRNA and tRNAs for almost all amino acids have been observed in the phloem sap of cucurbits42. They appear to be selectively transferred into the sieve tube system. The tRNAs show modifications necessary for the ability to transfer amino acids to the translational apparatus. Yet, rather than participating in translation, they appear to inhibit this process. Alternatively, they could serve as a source of cytokinin or signal the metabolic status of the plant42.</li>
	<li>Fatty acids, oxylipins, and other lipids have also been found in phloem exudates 3, 13, 14, 23. Jasmonic acid, an oxylipin moves through the phloem as part of the response to pathogen infection22, 29, 31, 32. While the role of most phloem lipids is still unclear, some of them likely have signaling function4.</li>
</ul>
The challenge in working with plant phloem lies in its capacity to seal itself upon wounding. There are four major methods used to collect phloem exudates, yet they work only in select species:
1) In cucurbits it is possible to obtain reasonable amounts of phloem exudate through cuts of the petiole. Once the initial drop with contamination of injured cells is removed, it is possible to obtain reasonably large amounts of pure phloem sap 1, 15. However, with increasing collection time, this exudate thickens, making it increasingly unsuitable for liquid chromatography approaches (unpublished). Recent publications suggest, that depending on the species, this phloem sap is derived from either the fascicular (FP) or the extrafascicular phloem (EP) and that, while it does contain &#34;mobile&#34; phloem sap from the sieve elements (FP), it is also prone to contamination from other cell types, including the xylem40, 41.
2) A second approach is to obtain phloem sap through shallow cuts or punctures in stem or petiole. This method has been used successfully in lupine 17, 25, cucurbits36, and Brassica napus12. Here the contamination from injured cells is minimal and the exudates very pure. However, plants need to be healthy and well-watered since it is very challenging to selectively puncture just the sieve elements. If xylem vessels are nicked, all exudate is drawn into the xylem stream. This makes the method unsuitable for plants with very fragile or highly lignified petioles or stems.
3) Aphid stylectomy allows aphids to insert their stylet into the sieve elements then removing the aphid with a laser. Phloem sap is exuded through the remaining stylet2, 9, 10, 35, 38. In theory, any plant that can be infected by aphids can be used for this approach. However, most greenhouse or growth chamber managers will not support use of a pathogen. In addition, aphids introduce several proteins into the phloem with their saliva 19, 33. This leads to a limited transcriptional reprogramming 30 and has the potential to change the phloem composition26.
4) The method described here is the EDTA-facilitated exudation of phloem sap. This method employs EDTA to prevent sealing of the phloem20. EDTA chelates Ca2+ ions that would otherwise participate in processes that seal the phloem. While EDTA can lead to cell damage30, several groups have used this method and observed no adverse effect of EDTA concentrations from 10 mM to 20 mM on the cell ultrastructure or on phloem loading and transport5, 24. To reduce any damaging effect as well as interference of EDTA with chromatography and gel electrophoresis, plants are moved into water after 1 h and only the later part of the exudate is used14. Thus, rather than exudation into EDTA, phloem is exudated into water (EDTA-facilitated exudation). It is a straight-forward, low cost, and low-tech method for the collection of phloem exudates. Phloem sap obtained this way can be used to analyze proteins, small molecules, lipids, and RNAs and has been used successfully in many plants. While a limited amount of experiments has been performed in monocots11, the method appears to be more suitable for dicots (Perilla17, 20, Arabidopsis8, 13, 14, Poplar7). Collection of exudates has to occur in a humid environment to avoid loss of exudates through transpiration. Depending on the plant, incubation in EDTA for one to two hours is sufficient to prevent sealing of the phloem/ sieve elements. The collection can then occur into water. This has the benefit of preventing the negative effect EDTA has on cell structure and stability. It also eliminates the interference of EDTA with methods like HPLC or SDS-Page. It is shown as it applies to Arabidopsis. For larger plants, incubation in EDTA and exudate collections has to be scaled up and is performed in beakers rather than in 1.7 ml reaction tubes.

<table border="0" cellpadding="0" cellspacing="0" width="638">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">K2-EDTA</td>
			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:111px;">ED2P-500G</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:153px;">Shallow Glass or plastic Petri Dish (7-15 cm)</td>
			<td>PYREX or Corning</td>
			<td></td>
			<td>Any clean, shallow dish will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloroform</td>
			<td>EMD</td>
			<td>CX1054-1</td>
			<td style="width:223px;">Only open containers in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td style="width:152px;">J.T.Baker</td>
			<td style="width:111px;">9070-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">Screw cap tubes</td>
			<td style="width:152px;">VWR International</td>
			<td style="width:111px;">53283-800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">Screw cap tubes</td>
			<td style="width:152px;">Sun Sri</td>
			<td style="width:111px;">13-425</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">Eppendorf tubes</td>
			<td style="width:152px;">Denville</td>
			<td style="width:111px;">C2170</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

It has become clear over the past several years that the complexity of the phloem sap may be the answer to the question of how plants send varying developmental and stress signals for optimal response. Using EDTA-facilitated phloem exudation may provide the opportunity to analyze phloem exudates from plants that are not suitable for other approaches but may be of economic or physiological interest.
This method allows for the harvest of enough phloem exudates to identify phloem-localized proteins and detect changes in their level in response to development or stress (Figure 3). As the figure shows, proteins are in very low abundance, yet, their level is high enough for subsequent proteomics experiments using LC-MS/MS or for Western blot analysis. Findings in cucurbits suggest that the cutting of the stem leads to a disruption of the water potential balance and a subsequent influx of water and possible contaminants from the apoplast41. Yet collection for times ranging from one to eight hours does not affect the phloem protein profile/composition in Arabidopsis (only the absolute amount varies Guelette et al.), the amount of protein collected and the pattern of proteins found varies between species (Arabidopsis: A.t.; Perilla, lane I and NI) and treatments (Perilla grown at different day lengths, lane I and NI). As a result proteinaceous signals can be visualized, identified, and followed.
mRNA and micro RNAs can also be identified in phloem exudates by this method. However, treatment with RNAse inhibitor is necessary to prevent degradation of the mRNA during the extended exudation time. Since the sieve elements do not contain functional chloroplasts, Rubisco small or large subunit (RbcS or RbcL, respectively) mRNAs can be used as negative controls, while known phloem-localized mRNA like Ubiquitin-conjugating enzyme may be used as a positive control (Figure 4).
Similarly, sugars or small metabolites can be analyzed using either GC-MS or LC-MS. Here it should be mentioned that the phloem exudates contain a large number of functional enzymes, including almost the entire glycolytic pathway12. Hence, using several time points may reveal metabolic processes during the exudate collection. An example is shown in Figure 5: In all exudates, sucrose is the most abundant metabolite; this is most obvious after a collection for 1 hour. However, after five hours the sucrose to fructose ratio is slightly reduced. If the same exudate is collected for one hour and left on the bench for the next four hours, the sucrose to fructose ratio is reduced to a much larger extent, suggesting that active enzymes in the phloem exudates lead to a degradation of sucrose at room temperature (for more peak interpretations see 14). This is consistent with findings that phloem loading and transport of molecules from companion cells into the sieve elements may continue during exudation until the system loses its vitality34.
Lipids in the phloem exudates and, by extension, long-distance lipid signaling are a fairly recent field of interest in plant science. Due to their hydrophobic nature lipids are only present in low concentrations and may be bound to other molecules for solubilization. Yet, the EDTA-facilitated exudation allows for the collection of sufficient material to visualize (TLC; Figure 6) and identify (LC-MS; Figure 7) phloem lipids from several plant species. As the figure shows it is possible to identify and separate several lipid species. LC-MS allows to identify different lipid species and to monitor changes within the lipid profiles of different genotypes or treatments. This allows for the study of the role of lipids during plant development and stress response.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51111/51111fig1.jpg" width="600px" /> 
Figure 1. Flow chart of the collection of phloem exudates of Arabidopsis or Perilla. Different paths lead to different end products, which are indicated in blue. For preparation of RNA, RNAse inhibitor (100 U/ml, Roche) is added to the water into which the phloem exudate is collected. 
<a href="https://www.jove.com/files/ftp_upload/51111/51111fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51111/51111fig2.jpg" width="600px" /> 
Figure 2. Setup of materials for phloem exudate collection. The setup displays all materials needed for protocol steps 3.1-3.4. 
<a href="https://www.jove.com/files/ftp_upload/51111/51111fig2highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51111/51111fig3.jpg" width="600px" /> 
Figure 3. Proteins found in phloem exudates of two different plant species, Arabidopsis (A.t.) and Perilla (I and NI; different day lengths); MW: molecular weight marker (lane 2). Proteins were separated using a 10-20% gradient SDS-PAGE. 
<a href="https://www.jove.com/files/ftp_upload/51111/51111fig3highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51111/51111fig4.jpg" width="600px" /> 
Figure 4. Analysis of the presence of mRNA for Rubisco small and large subunit (RbcS and RbcL, respectively) and ubiquitin-conjugating enzyme (UBC). mRNA was collected from Arabidopsis leaves (L), petioles (P), and phloem exudates (Ph), reverse transcribed and specific transcripts visualized using PCR. The figure was modified from Guelette et al.14. 
<a href="https://www.jove.com/files/ftp_upload/51111/51111fig4highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/51111/51111fig5.jpg" width="600px" /> 
Figure 5. GC-MS profile of phloem exudates collected for different amount of times. Note how the relative amount of sucrose changes from 1 hour collection (A) to 5 hours of collection time (B). Exudate profile after collection for 1 hour followed by &#34;incubation&#34; at room temperature (RT) for four hours (C). Leaf metabolite profile (D). 
<a href="https://www.jove.com/files/ftp_upload/51111/51111fig5highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 6" fo:content-width="5in" src="/files/ftp_upload/51111/51111fig6.jpg" width="600px" /> 
Figure 6. Thin-layer chromatogram comparing lipids from Perilla (left) and Arabidopsis (right) leaves and phloem exudates. Asterisks indicate lipids specific for phloem exudates. DGDG: digalactosyldiacylglycerol; PG: phosphatidylglycerol; MGDG: monogalactosyldiacyglycerol; The part of the figure displaying Arabidopsis lipids has been modified from Guelette et al.14. 
<a href="https://www.jove.com/files/ftp_upload/51111/51111fig6highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 7" fo:content-width="5in" src="/files/ftp_upload/51111/51111fig7.jpg" width="600px" /> 
Figure 7. Lipid profile of phloem exudates of Arabidopsis (chloroform phase) using LC-MS/ negative ion mode. Graphs in the top (mutant, A) and bottom (wild-type, B) show the differences in the lipid profiles between two different genotypes (indicated by arrows). 
<a href="https://www.jove.com/files/ftp_upload/51111/51111fig7highres.jpg" target="_blank">Click here to view larger image</a>.

Watch this Scientific Journal Video about Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method at JoVE.com

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.

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 ...

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24.3K Views.  Michigan State Universtiy. 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.

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

Collection of Alfalfa Root Exudates to Study the Impact of Di(2-ethylhexyl) Phthalate on Metabolite Production

Root exudates are the main media of information communication and energy transfer between plant roots and the surrounding environment. The change in secretion of root exudates is usually an external detoxification strategy for plants under stress conditions. This protocol aims to introduce general guidelines for the collection of alfalfa root exudates to study the impact of di(2-ethylhexyl) phthalate (DEHP) on metabolite production. First, alfalfa seedlings are grown under DEHP stress in a hydroponic culture experiment. Second, the plants are transferred to centrifuge tubes containing 50 mL of sterilized ultrapure water for 6 h to collect root exudates. The solutions are then freeze-dried in a vacuum freeze dryer. The frozen samples are extracted and derivatized with bis(trimethylsilyl)) trifluoroacetamide (BSTFA) reagent. Subsequently, the derivatized extracts are measured using a gas chromatograph system coupled with a time-of-flight mass spectrometer (GC-TOF-MS). The acquired metabolite data are then analyzed based on bioinformatic methods. Differential metabolites and significantly changed metabolism pathways should be deeply explored to reveal the impact of DEHP on alfalfa in view of root exudates.

Collection of Alfalfa Root Exudates to Study the Impact of Di2-ethylhexyl Phthalate on Metabolite Production

The secretion of root exudates is usually an external detoxification strategy for plants under stress conditions. This protocol describes how to assess the impact of xenobiotics on alfalfa via nontargeted metabolomic analysis.

Root exudates are the main media of information communication and energy transfer between plant roots and the surrounding environment. The change in ...

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Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based phosphoproteomics has transformed the way of studying plant signal transduction. However, requirement of lots of starting materials and prolonged MS measurement time to achieve the depth of coverage has been the limiting factor for the high throughput study of global phosphoproteomic changes in plants. To improve the sensitivity and throughput of plant phosphoproteomics, we have developed a stop and go extraction (stage) tip based phosphoproteomics approach coupled with Tandem Mass Tag (TMT) labeling for the rapid and comprehensive analysis of plant phosphorylation perturbation in response to osmotic stress. Leveraging the simplicity and high throughput of stage tip technique, the whole procedure takes approximately one hour using two tips to finish phosphopeptide enrichment, fractionation, and sample cleaning steps, suggesting an easy-to-use and high efficiency of the approach. This approach not only provides an in-depth plant phosphoproteomics analysis (&#62; 11,000 phosphopeptide identification) but also demonstrates the superior separation efficiency (&#60; 5% overlap) between adjacent fractions. Further, multiplexing has been achieved using TMT labeling to quantify the phosphoproteomic changes of wild-type and snrk2 decuple mutant plants. This approach has successfully been used to reveal the phosphorylation events of Raf-like kinases in response to osmotic stress, which sheds light on the understanding of early osmotic signaling in land plants.

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Presented here is a phosphoproteomic approach, namely stop and go extraction tip based phosphoproteomic, which provides high-throughput and deep coverage of Arabidopsis phosphoproteome. This approach delineates the overview of osmotic stress signaling in Arabidopsis.

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based ...

Biochemistry

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 ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

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

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

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

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

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

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

Phloem Sap Sampling from Brassica napus for 3D-PAGE of Protein and Ribonucleoprotein Complexes

Sampling the phloem of higher plants is often laborious and significantly dependent on the plant species. However, proteome studies under denaturing conditions could be achieved in different plant species. Native protein:protein and protein:nucleic acid complexes from phloem samples have as yet scarcely been analyzed, although they might play important roles in maintenance of this specialized compartment or in long-distance signaling. Large molecular assemblies can be isolated using a blue native gel electrophoresis (BN-PAGE). Their protein components can be separated by a subsequent sodium dodecyl sulfate PAGE (SDS-PAGE). However, proteins with similar molecular weights co-migrate, what can hinder protein identification by mass spectrometry. Combining BN-PAGE with two different denaturing gel electrophoresis steps, namely Tris-Tricine-urea and SDS-PAGE, enables the additional separation of proteins according to their hydrophilicity/hydrophobicity and thus increases resolution and the success of protein identification. It even allows distinguishing proteins that only differ in their posttranslational modifications. In addition, blue native northern blotting can be applied to identify the RNA components in macromolecular complexes. We show that our protocol is suitable to unravel the protein and RNA components of native protein:protein and ribonucleoprotein (RNP) complexes occurring in phloem samples. Combining a blue native PAGE with two different denaturing PAGE steps can help to separate different kinds of large protein complexes, and also enables an increased identification rate of their components by mass spectrometry. Furthermore, the protocol is robust enough to simultaneously detect potentially bound nucleic acids within single protein complexes.

Phloem Sap Sampling from Brassica napus for 3D-PAGE of Protein and Ribonucleoprotein Complexes

Here we present a protocol to analyze the protein composition of large native protein:protein and protein:nucleic acid complexes from oilseed rape (B. napus) phloem exudate using a 3D polyacrylamide gel electrophoresis (PAGE) approach combining blue native (BN) with two denaturing PAGEs followed by mass spectrometric identification.

Sampling the phloem of higher plants is often laborious and significantly dependent on the plant species. However, proteome studies under denaturing ...

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 ...

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.

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 ...

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 ...