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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24.2K 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

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

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 ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently attach a tagged form of dUTP to 3&#8217; ends of double- and single-stranded DNA breaks in cells. It is a reliable and useful method to detect DNA damage and cell death in situ. This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method of semi-automated TUNEL signal quantitation. Inherent normal tissue features and tissue processing conditions affect the ability of the TdT enzyme to efficiently label DNA. Tissue processing may also add undesirable autofluorescence that will interfere with TUNEL signal detection. Therefore, it is important to empirically determine tissue processing and TUNEL labeling methods that will yield the optimal signal-to-noise ratio for subsequent quantitation. The fluorescence-based assay described here provides a way to exclude autofluorescent signal by digital channel subtraction. The TUNEL assay, used with appropriate tissue processing techniques and controls, is a relatively fast, reproducible, quantitative method for detecting apoptosis in tissue. It can be used to confirm DNA damage and apoptosis as pathological mechanisms, to identify affected cell types, and to assess the efficacy of therapeutic treatments in vivo.

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method for semi-automated analysis of TUNEL labeling.

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...

Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection

Rice stripe virus (RSV), which causes significant economic loss of agriculture in East Asia, entirely depends on insect vectors for its effective transmission among host rice. Laodelphax striatellus (small brown planthopper, SBPH) is the primary insect vector that horizontally transmits RSV while sucking sap from the phloem. Saliva plays a significant role in insects' feeding behavior. A convenient method that will be useful for research on insects' saliva with piercing-sucking feeding behavior is described here. In this method, insects were allowed to feed on an artificial diet sandwiched between two stretched paraffin film layers. The diet containing the saliva was collected each day, filtered, and concentrated for further analysis. Finally, the quality of collected saliva was examined by protein staining and immunoblotting. This method was exemplified by detecting the presence of RSV and a mucin-like protein in the saliva of SBPH. These artificial feeding and saliva collection method will lay a foundation for further research on factors in insect saliva related to feeding behavior and virus transmission.

Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection

The present protocol describes a method to collect sufficient saliva from piercing-sucking insects using an artificial medium. This is a convenient method for collecting insect saliva and studying salivary function on insect feeding behavior and vector-borne virus transmission.

Rice stripe virus (RSV), which causes significant economic loss of agriculture in East Asia, entirely depends on insect vectors for its effective ...

High-throughput, Robust and Highly Time-flexible Method for Surface Sterilization of Arabidopsis Seeds

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental step required towards this end. Thus, it is paramount to establish high-throughput Arabidopsis seed surface sterilization methods to handle tens to hundreds of samples (e.g., transgenic lines, ecotypes, or mutants) at once. A seed surface sterilization method based on the efficient elimination of liquid in tubes with a homemade suction device constructed from a common vacuum pump is presented in this study. By dramatically reducing labor-intensive hands-on time with this method handling several hundreds of samples in one day is possible with little effort. Series time-course analyses further indicated a highly flexible time range of surface sterilization by maintaining high germination rates. This method could be easily adapted for surface sterilization of other kinds of small seeds with simple customization of the suction device according to the seed size, and the speed desired to eliminate the liquid.

A high-throughput protocol for the surface sterilization of Arabidopsisthaliana (Arabidopsis) seeds is provided, optimizing the liquid handling steps with a simple suction device constructed with a vacuum pump. Hundreds of seed samples can be surface-sterilized in one day.

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...

Live Imaging of Arabidopsis Pollen Tube Reception and Double Fertilization Using the Semi-In Vitro Cum Septum Method

In flowering plants, the growth and guidance of the pollen tube (male gametophyte) within the pistil and the reception of the pollen tube by the female gametophyte are essential for double fertilization and subsequent seed development. The interactions between male and female gametophytes during pollen tube reception culminate in pollen tube rupture and the release of two sperm cells to effect double fertilization. As pollen tube growth and double fertilization are deeply hidden within the tissues of the flower, this process is difficult to observe in vivo.
A semi-in vitro (SIV) method for the live-cell imaging of fertilization in the model plant Arabidopsis thaliana has been developed and implemented in several investigations. These studies have helped to elucidate the fundamental features of how the fertilization process occurs in flowering plants and which cellular and molecular changes occur during the interaction of the male and female gametophytes. However, because these live cell imaging experiments involve the excision of individual ovules, they are limited to a low number of observations per imaging session, making this approach tedious and very time-consuming. Among other technical difficulties, a failure of the pollen tubes to fertilize the ovules in vitro is&#160;often reported, which severely confounds such analyses.
Here, a detailed video protocol for the imaging of pollen tube reception and fertilization in an automated and high-throughput manner is provided, allowing for up to 40 observations of pollen tube reception and rupture per imaging session. Coupled with the use of genetically encoded biosensors and marker lines, this method enables the generation of large sample sizes with a reduced time investment. Nuances and critical points of the technique, including flower staging, dissection, medium preparation, and imaging, are clearly detailed in video format to facilitate future research on the dynamics of pollen tube guidance, reception, and double fertilization.

Live Imaging of Arabidopsis Pollen Tube Reception and Double Fertilization Using the Semi-In Vitro Cum Septum Method

Here, we describe an improvement of the semi-in vitro (SIV) method for observing pollen tube guidance and reception in Arabidopsis thaliana,&#160;which increases the receptivity of ovules. The high-throughput SIV cum septum method may be coupled with gametophyte marker lines and genetically encoded biosensors to monitor the dynamic process of fertilization.

In flowering plants, the growth and guidance of the pollen tube (male gametophyte) within the pistil and the reception of the pollen tube by the female ...