Name: the infiltration-centrifugation technique for extraction of apoplastic fluid from plant leaves using phaseolus vulgaris as an example
Uploaded: 2014-12-19T13:00:00
Duration: 10 min 26 s
Description: Watch this Scientific Journal Video about The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example at JoVE.com

This work was supported by grants BB/J016012/1 and BB/E007872/1 from the UK Biotechnology and Biological Sciences Research Council (BBSRC) to Gail Preston.

NOTE: The choice of starting leaf material and standardization of plant growth conditions is critical as these factors can greatly affect the quality, variability and yield of AWF (Figure 1). Parameters to consider are shown in Table 1 and are discussed in more detail in the discussion.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Parameter</td>
			<td colspan="3">Example standardizations</td>
		</tr>
		<tr>
			<td></td>
			<td>Phaseolus vulgaris</td>
			<td>Solanum lycopersicum</td>
			<td>Arabidopsis thaliana</td>
		</tr>
		<tr>
			<td>Leaf type</td>
			<td>First true leaves, fully expanded, healthy</td>
			<td>2nd and 3rd leaves starting from bottom, 4 largest leaflets taken apical leaflet not used for apoplast extraction</td>
			<td>Fully expanded rosette leaves</td>
		</tr>
		<tr>
			<td>Plant age</td>
			<td>21 days</td>
			<td>7 - 9 weeks</td>
			<td>7 weeks&#160;</td>
		</tr>
		<tr>
			<td>Time of day</td>
			<td>Middle of light period (12:00)</td>
			<td>Middle of light period (12:00)</td>
			<td>Middle of light period (12:00)</td>
		</tr>
		<tr>
			<td>Hydration</td>
			<td>All plants well watered 1 hr pre-harvest</td>
			<td>All plants well watered 1 hr pre-harvest</td>
			<td>All plants well watered 1 hr pre-harvest</td>
		</tr>
		<tr>
			<td>Light</td>
			<td>16 hr light, 400 &#181;mol m-2 sec-1</td>
			<td>16 hr light, 400 &#181;mol m-2 sec-1</td>
			<td>10 hr light (short day) from week 2, 400 &#181;mol m-2 sec-1</td>
		</tr>
		<tr>
			<td>Humidity</td>
			<td>70%</td>
			<td>70%</td>
			<td>70%</td>
		</tr>
		<tr>
			<td>Temperature</td>
			<td>22 &#176;C light, 18 &#176;C dark</td>
			<td>22 &#176;C light, 18 &#176;C dark</td>
			<td>21 &#176;C</td>
		</tr>
	</tbody>
</table>
Table 1. Examples of standardized parameters for leaves used in AWF extractions.
1. Generating Apoplast Washing Fluid
NOTE: During this protocol hazardous materila inculding microbial pathogens from infected leaves should not be washed down the drain; rather proper disposal procedures should be used.
<ol>
	<li>Choose apparatus based on leaf size:
	<ol>
		<li>Infiltrate smaller leaves (e.g., Arabidopsis, bean) using a needleless syringe. Use a vacuum flask to infiltrate leaves that are too large to fit in a syringe. Use the vacuum flask method to infiltrate multiple leaves simultaneously.</li>
		<li>Divide leaves that are too large for the available infiltration method into smaller sections using a razor blade. Thoroughly rinse cut leaf sections in distilled water prior to infiltration to remove cytoplasmic contaminants that exude from damaged cells.</li>
	</ol>
	</li>
	<li>Leaf infiltration using a 60 ml syringe:
	<ol>
		<li>Detach the leaf from the plant at the petiole using a razor blade. For compound leaves such as tomato, detach leaflets at the petiolule.</li>
		<li>Rinse the freshly excised leaf by submersion in distilled water to remove leaf surface contaminants. Dry the leaf by gently blotting with absorbent tissue or paper.</li>
		<li>Measure the leaf weight before infiltration.</li>
		<li>Carefully roll and/or fold the leaf into a 60 ml syringe and fill the syringe with distilled water (other infiltration fluids may be used, see discussion). Eject any air within the syringe while lowering the plunger to approximately the 40 ml mark.</li>
		<li>Cover the syringe tip with either a gloved finger or a piece of Parafilm, then create negative pressure within the syringe by pulling the plunger outwards to the 60 ml mark. Slowly release the plunger. 
		NOTE: Quickly releasing the pressure may result in cell lysis and cytoplasmic contamination.</li>
		<li>Unplug the syringe tip and eject any air, then replug the tip and press further downwards on the plunger to create a modest amount of positive pressure.</li>
		<li>Repeat steps 1.2.5 - 1.2.6 until the leaf is fully infiltrated, as seen by the darkened colour of the infiltrated areas.</li>
		<li>Remove the leaf from the syringe. Gently but thoroughly blot the leaf with absorbent paper to remove surface liquid.</li>
		<li>Measure the weight of the infiltrated leaf. Calculate the difference in weight before and after infiltration which closely approximates the infiltration volume.</li>
	</ol>
	</li>
	<li>Leaf infiltration by vacuum flask:
	<ol>
		<li>Detach the leaf from the plant at the petiole using a razor blade.</li>
		<li>Rinse the freshly excised leaf by submersion in distilled water to remove leaf surface contaminants. Dry the leaf by gently blotting with absorbent paper.</li>
		<li>Measure the leaf weight before infiltration.</li>
		<li>Place the leaf (or leaves if infiltrating multiple leaves) in a 250 ml or larger side arm flask containing distilled water (other infiltration fluids may be used, see Discussion). Fully cover the leaves with the liquid.</li>
		<li>Apply a vacuum to the flask. Gently agitate to release the air bubbles from the leaves. Carefully and slowly release the vacuum. 
		NOTE: Quickly releasing the vacuum may result in cell lysis and cytoplasmic contamination.</li>
		<li>Repeat step 1.3.5 until the leaf is fully infiltrated, as seen by the darkened colour of the infiltrated areas.</li>
		<li>Remove the leaf from the flask. Gently but thoroughly blot the leaf with absorbent paper to remove surface liquid.</li>
		<li>Measure the weight of the infiltrated leaf. Calculate the difference in weight before and after infiltration which closely approximates the infiltration volume.</li>
	</ol>
	</li>
	<li>Recovery of AWF by centrifugation:
	<ol>
		<li>Place the leaf on a piece of 4 inch (10 cm) wide Parafilm. Using a 5 ml pipette tip (or a similar sized object), roll up the leaf within the Parafilm.</li>
		<li>Insert the rolled up leaf with pipette tip into a 20 ml syringe. Position any cut leaf edges facing upwards. Insert the syringe into a clean 50 ml polyethylene or polycarbonate tube.</li>
		<li>Centrifuge the syringe within the tube for 10 min at 1,000 x g in a swinging bucket rotor at 4 &#176;C. 
		NOTE: Visual examination of the leaves following centrifugation should reveal that the majority of the infiltration fluid has been expelled. Darker green patches indicate that additional centrifugation time is required.</li>
		<li>Pipette the recovered AWF into fresh 1.5 ml tubes. 
		NOTE: The volume of AWF should approximately match the infiltration volume for that leaf; if the volume is less then additional centrifugation time or centrifugation speed is required.</li>
		<li>Centrifuge the AWF samples at 15,000 x g for 5 min to remove any cells or particulate matter. Alternatively, pass the AWF through a 0.22 &#181;m filter to remove cells and particulate matter.</li>
		<li>Pipette the supernatant into a fresh tube. Keep samples on ice until storage.</li>
		<li>Store the AWF samples at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Assays for Cytoplasmic Contamination
<ol>
	<li>Keep the AWF samples on ice to minimize enzymatic reactions (e.g., proteolysis) and perform the assays immediately after the centrifugation steps are complete. 
	NOTE: For metabolite assays, samples may be immediately frozen after extraction and stored at -80 &#176;C until use.</li>
	<li>For a standard malate dehydrogenase (MDH) assay protocol see 15; otherwise use a commercially available MDH assay kit.</li>
	<li>For a standard glucose-6-phosphate dehydrogenase (G6PDH) assay protocol see 16; otherwise use a commercially available G6PDH assay kit.</li>
	<li>For quantification of cytoplasmic metabolites such as glucose-6-phosphate (G-6-P) use GC-MS as described in step 5. Otherwise, use a commercially available kit for the direct assay of G-6-P.</li>
</ol>
3. Calculation of the Apoplast Dilution Factor
<ol>
	<li>Prepare two volumes of the infiltration fluid used above in step 1.2.4 or 1.3.4 (e.g., distilled water). Add indigo carmine powder to one solution to a final concentration of 50 &#181;M, add nothing to the other.</li>
	<li>Measure the absorbance at 610 nm (OD610) of 200 &#181;l of the indigo carmine infiltration solution with a plate reader. Correct this value by subtracting the OD610 of 200 &#181;l of infiltration solution without indigo carmine. Substitute this corrected value as OD610infiltrate into the equation below.</li>
	<li>Perform at least three replicate leaf infiltrations as above (step 1.2 or 1.3) with both infiltration solutions (with and without indigo carmine).</li>
	<li>After infiltration, rinse the leaves in distilled water to remove any remaining indigo carmine present on the outer surfaces.</li>
	<li>Proceed with centrifugation as above (step 1.4).</li>
	<li>Determine the average OD610 of 200 &#181;l of the indigo carmine AWF extractions. Correct this value by subtracting the average OD610 value of 200 &#181;l of indigo carmine free AWF. Substitute this corrected value as OD610AWF into the equation below.</li>
	<li>Calculate the dilution factor (i.e., fold dilution) from the corrected absorbance values as: 
	Apoplast dilution factor = OD610infiltrate / (OD610infiltrate - OD610AWF)</li>
	<li>Multiply concentration or activity values from AWF by the dilution factor to estimate the concentration/activity in apoplastic fluid in planta.</li>
</ol>
4. Concentration of AWF to Full Strength by Freeze Drying
<ol>
	<li>Turn on the freeze-drier and allow it to stabilize at the working temperature and pressure.</li>
	<li>Pool the desired amount of fresh or stored AWF samples.</li>
	<li>Distribute the samples evenly among 2 ml centrifuge tubes.</li>
	<li>Determine the reconstitution volume: 
	Reconstitution volume = volume before freeze-drying / apoplast dilution factor</li>
	<li>With the lids closed, freeze the tubes in liquid nitrogen.</li>
	<li>Transfer the frozen tubes to one or more chilled freeze-drying vessels. While transferring the tubes, replace the lid with one that has been pierced with a sharp object to allow gas exchange.</li>
	<li>Attach the vessels to the freeze-drier and lyophilize the samples completely.</li>
	<li>Remove the samples from the machine and reconstitute the AWF by adding the calculated amount of distilled water.</li>
	<li>Replace the unpierced lid and store the samples at -80 &#176;C.</li>
</ol>
5. Metabolite Analysis by Gas Chromatography Mass Spectrometry17
<ol>
	<li>Pipette 200 &#181;l of AWF into a 1.5 ml tube</li>
	<li>Add 700 &#181;l of methanol containing 10 &#181;g/ml ribitol as an internal standard.</li>
	<li>Heat at 70 &#176;C for 10 min.</li>
	<li>Centrifuge for 10 min at 11,000 x g.</li>
	<li>Transfer 700 &#181;l of supernatant to a fresh 1.5 ml tube.</li>
	<li>Add 375 &#181;l of cold chloroform and vortex for 10 sec.</li>
	<li>Add 500 &#181;l of dH2O and vortex for 10 sec.</li>
	<li>Centrifuge for 15 min at 2,200 x g.</li>
	<li>Transfer 150 &#181;l of the upper aqueous layer into a new 1.5 ml tube, then dry the samples completely in a vacuum concentrator without heat.</li>
	<li>Dissolve the samples in 30 &#181;l of pyridine containing 20 mg/ml methoxyamine hydrochloride.</li>
	<li>Incubate at 70 &#176;C while shaking vigorously for 2 hr.</li>
	<li>Add 50 &#181;l of MSTFA (N-methyl-N-(trimethylsilyl) trifluoroacetamide).</li>
	<li>Incubate at 37 &#176;C while shaking for 30 min.</li>
	<li>Transfer samples to GC-MS compatible glass vials.</li>
	<li>Perform GC-MS analysis of the samples. Refer to Lisec et al. 200917, for details on GC-MS equipment setup and performance.</li>
</ol>

<ol>
	<li>Felle, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoplastic+pH+during+low-oxygen+stress+in+barley.">Apoplastic pH during low-oxygen stress in barley.</a> Annals of Botany. 98 (5), 1085-1093 (2006).</li><li>Schaarschmidt, S., Kopka, J., Ludwig-M&#252;ller, J., Hause, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+arbuscular+mycorrhization+by+apoplastic+invertases:+enhanced+invertase+activity+in+the+leaf+apoplast+affects+the+symbiotic+interaction.">Regulation of arbuscular mycorrhization by apoplastic invertases: enhanced invertase activity in the leaf apoplast affects the symbiotic interaction.</a> The Plant Journal. 51 (3), 390-405 (2007).</li><li>Outlaw, W. H., De Vlieghere-He, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transpiration+rate.+An+important+factor+controlling+the+sucrose+content+of+the+guard+cell+apoplast+of+broad+bean.">Transpiration rate. An important factor controlling the sucrose content of the guard cell apoplast of broad bean.</a> Plant Physiology. 126 (4), 1716-1724 (2001).</li><li>Rico, A., Jones, R., Preston, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+to+the+plant+apoplast+by+plant+pathogenic+bacteria.">Adaptation to the plant apoplast by plant pathogenic bacteria.</a> Plant Pathogenic Bacteria. , 63-89 (2009).</li><li>Rico, A., Preston, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+syringae+pv.+tomato+DC3000+uses+constitutive+and+apoplast-induced+nutrient+assimilation+pathways+to+catabolize+nutrients+that+are+abundant+in+the+tomato+apoplast.">Pseudomonas syringae pv. tomato DC3000 uses constitutive and apoplast-induced nutrient assimilation pathways to catabolize nutrients that are abundant in the tomato apoplast.</a> Molecular Plant-Microbe Interactions. 21 (2), 269-282 (2008).</li><li>Solomon, P., Tan, K., Oliver, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nutrient+supply+of+pathogenic+fungi;+a+fertile+field+for+study.">The nutrient supply of pathogenic fungi; a fertile field for study.</a> Molecular Plant Pathology. 4, 203-210 (2003).</li><li>Brunner, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immunity+in+plants+and+animals:+emerging+parallels+between+the+recognition+of+general+elicitors+and+pathogen-associated+molecular+patterns.">Innate immunity in plants and animals: emerging parallels between the recognition of general elicitors and pathogen-associated molecular patterns.</a> Current Opinion in Plant Biology. 5 (4), 318-324 (2002).</li><li>Fontaniella, B., Vicente, C., Armas, R., Legaz, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+leaf+scald+(Xanthomonas+albilineans)+on+polyamine+and+phenolic+acid+metabolism+of+two+sugarcane+cultivars.">Effect of leaf scald (Xanthomonas albilineans) on polyamine and phenolic acid metabolism of two sugarcane cultivars.</a> European Journal of Plant Pathology. 119 (4), 401-409 (2007).</li><li>Mill&#225;n, A. F., Morales, F., Abad&#237;a, A., Abad&#237;a, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+iron+deficiency+on+the+composition+of+the+leaf+apoplastic+fluid+and+xylem+sap+in+sugar+beet.+Implications+for+iron+and+carbon+transport.">Effects of iron deficiency on the composition of the leaf apoplastic fluid and xylem sap in sugar beet. Implications for iron and carbon transport.</a> Plant Physiology. 124 (2), 873-884 (2000).</li><li>Dragi&#353;i&#263; Maksimovi&#263;, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filter+strip+as+a+method+of+choice+for+apoplastic+fluid+extraction+from+maize+roots.">Filter strip as a method of choice for apoplastic fluid extraction from maize roots.</a> Plant Science. 223 (1), 49-58 (2014).</li><li>Baker, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+internal+standard+technique+for+improved+quantitative+analysis+of+apoplastic+metabolites+in+tomato+leaves.">An internal standard technique for improved quantitative analysis of apoplastic metabolites in tomato leaves.</a> Physiological and Molecular Plant Pathology. 78, 31-37 (2012).</li><li>Nouchi, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overcoming+the+difficulties+in+collecting+apoplastic+fluid+from+rice+leaves+by+the+infiltration-centrifugation+method.">Overcoming the difficulties in collecting apoplastic fluid from rice leaves by the infiltration-centrifugation method.</a> Plant &amp; Cell Physiology. 53 (9), 1659-1668 (2012).</li><li>Boudart, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+wall+proteins+in+apoplastic+fluids+of+Arabidopsis+thaliana+rosettes:+identification+by+mass+spectrometry+and+bioinformatics.">Cell wall proteins in apoplastic fluids of Arabidopsis thaliana rosettes: identification by mass spectrometry and bioinformatics.</a> Proteomics. 5 (1), 212-221 (2005).</li><li>Lohaus, G., Pennewiss, K., Sattelmacher, B., Hussmann, M., Hermann Muehling, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+the+infiltration-centrifugation+technique+appropriate+for+the+isolation+of+apoplastic+fluid?+A+critical+evaluation+with+different+plant+species.">Is the infiltration-centrifugation technique appropriate for the isolation of apoplastic fluid? A critical evaluation with different plant species.</a> Physiologia Plantarum. 111 (4), 457-465 (2001).</li><li>Bergmeyer, H. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malate+Dehydrogenase.">Malate Dehydrogenase.</a> Methods of Enzymatic Analysis. 2, 614 (1974).</li><li>Lohr, G. W., Waller, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose-6-phosphate+Dehydrogenase.">Glucose-6-phosphate Dehydrogenase.</a> Methods of Enzymatic Analysis. 2, 636-643 (1974).</li><li>Lisec, J., Schauer, N., Kopka, J., Willmitzer, L., Fernie, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas+chromatography+mass+spectrometry-based+metabolite+profiling+in+plants.">Gas chromatography mass spectrometry-based metabolite profiling in plants.</a> Nature Protocols. 1 (1), 387-396 (2006).</li><li>Li, Z. C., McClure, J. W., Hagerman, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+and+bound+apoplastic+activity+for+peroxidase,+beta-d-glucosidase,+malate+dehydrogenase,+and+nonspecific+arylesterase,+in+barley+(Hordeum+vulgare+L.)+and+oat+(Avena+sativa+L.)+primary+leaves.">Soluble and bound apoplastic activity for peroxidase, beta-d-glucosidase, malate dehydrogenase, and nonspecific arylesterase, in barley (Hordeum vulgare L.) and oat (Avena sativa L.) primary leaves.</a> Plant Physiology. 90 (1), 185-190 (1989).</li><li>Dani, V., Simon, W. J., Duranti, M., Croy, R. R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+tobacco+leaf+apoplast+proteome+in+response+to+salt+stress.">Changes in the tobacco leaf apoplast proteome in response to salt stress.</a> Proteomics. 5 (3), 737-745 (2005).</li><li>Shabab, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fungal+effector+protein+AVR2+targets+diversifying+defense-related+Cys+proteases+of+tomato.">Fungal effector protein AVR2 targets diversifying defense-related Cys proteases of tomato.</a> Plant Cell. 20 (4), 1169-1183 (2008).</li><li>Cosgrove, D. J., Cleland, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solutes+in+the+free+space+of+growing+stem+tissues.">Solutes in the free space of growing stem tissues.</a> Plant Physiology. 72 (2), 326-331 (1983).</li><li>Husted, S., Schjoerring, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoplastic+pH+and+ammonium+concentration+in+leaves+of+Brassica+napus.">Apoplastic pH and ammonium concentration in leaves of Brassica napus.</a> L. Plant Physiology. 109 (4), 1453-1460 (1995).</li><li>Kettle, A. J., Clark, B. M., Winterbourn, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+converts+indigo+carmine+to+isatin+sulfonic+acid.">Superoxide converts indigo carmine to isatin sulfonic acid.</a> The Journal of Biological Chemistry. 279 (18), 18521-18525 (2004).</li><li>Bournonville, C. F. G., D&#237;az-Ricci, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+determination+of+superoxide+in+plant+leaves+using+a+modified+NBT+staining+method.">Quantitative determination of superoxide in plant leaves using a modified NBT staining method.</a> Phytochemical Analysis. 22 (3), 268-271 (2011).</li><li>Speer, M., Kaiser, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+relations+of+symplastic+and+apoplastic+space+in+leaves+from+Spinacia+oleracea+L.+and+Pisum+sativum+L.+under+salinity.">Ion relations of symplastic and apoplastic space in leaves from Spinacia oleracea L. and Pisum sativum L. under salinity.</a> Plant Physiology. 97 (3), 990-997 (1991).</li></ol>

No conflicts of interest declared.

Optimizing the plant tissue source
Biological and technical variation can be large when performing apoplast extractions, thus a highly standardized workflow is helpful to increase continuity across experiments (Figure 1). Importantly, the source of plant tissue must be standardized, including leaf type, leaf age, growth/environmental conditions and time of day (Table 1). Large differences exist in the ease with which different leaves are infiltrated and the AWF subsequently recovered by centrifugation; these differences are correlated with stomata number, aperture size and mesophyll resistance12,14. Even among different cultivars of P. vulgaris there are large differences in the ease and yield of the AWF extraction procedure; for example leaves of the Tendergreen variety used here are more amenable to AWF extraction using this method than the Canadian Wonder cultivar. Within P. vulgaris the first true leaves are the biggest and the easiest to infiltrate, making them the obvious choice for apoplastic extractions. The apoplastic air and water volume has been shown to vary with leaf age in several species leading to differences in AWF extractability12,14. In P. vulgaris, older leaves become substantially more difficult to infiltrate and yield less AWF upon centrifugation; therefore leaves were harvested when they reached full expansion. Leaves which are difficult to infiltrate subsequently require higher centrifugation speeds to recover the AWF. One should therefore carefully screen several different leaf types and varieties before deciding upon a tissue source for large scale AWF extractions.
Plant growth conditions must also be standardized as much as possible within the context of the experiment. The use of growth cabinets is preferable as they allow consistent humidity, temperature and light intensity regiments to be maintained. The harvesting of leaves should always occur at the same time of day because the concentrations of metabolites, enzymes, etc. vary throughout the diurnal cycle14. Finally, to ensure that the plant leaves all have similar turgor pressure, the plants should be watered soon before (~1 hr) the harvest.
Optimization of leaf infiltration and centrifugation
Undesirable cytoplasmic contamination of the AWF due to partial cell lysis will result if the mechanical stress encountered by the cells is too high during the infiltration or centrifugation steps. Therefore, the procedure for a given leaf type should be an optimized trade-off between maximizing yield and minimizing cytoplasmic contamination of the AWF. In all cases, one should use the lowest centrifugation speed at which AWF can be recovered to avoid possible mechanical disruption of the leaf cells. Optimization of the centrifugation speed should be determined empirically for each leaf type by monitoring the volume recovered and apoplast contamination over a range of centrifugation speeds. It has been observed that marker enzyme activities, such as MDH, G6PDH and glucose-phosphate isomerase, remain low until a threshold centrifugal force is surpassed, above which these activities increase rapidly, presumably due to cytoplasmic leakage9,12,14. The use of Parafilm for support during the centrifugation step, as noted by Baker et al. 201211, can improve the efficacy of the AWF extraction and may minimize mechanical damage to the leaf blade caused by excessive folding and compression. Furthermore, the use of Parafilm improves visualization of the leaf after centrifugation when one should examine the leaf for damage and completeness of the AWF extraction.
Nouchi12 describe the optimization of AWF recovery from cut rice leaf sections, which are difficult to infiltrate and require higher centrifugation speeds to collect AWF because of their small stomatal openings. Improved wetting of the rice leaf surface, either by presoaking the leaves in distilled water or the addition of a surfactant to the infiltration fluid, facilitated the infiltration process. A higher centrifugation speed (6,000 x g) was also used while monitoring for apoplastic contamination12. When using cut leaf sections there is always the risk that cytoplasmic contamination will be more prevalent even with extensive washing of the wound sites; cut leaves should therefore only be used when necessary.
For many studies distilled water is used as the infiltration fluid11. However, compounds may be added to the infiltration fluid, such as salts or buffers, to improve the extraction of certain apoplastic compounds, especially proteins13. Lohaus14 evaluated the effect of ionic and osmotic strength on the composition of the recovered AWF and found them to be negligible. Changes in pH of the infiltration medium, however, may affect the AWF composition14.
Proper handling and storage of the extracted apoplastic fluid is important. AWF has been shown to contain an abundance of proteases and other enzymes13,20, as well as volatile organic compounds. Therefore, to reduce changes to the composition of AWF after recovery it is recommended to keep samples on ice or otherwise stored at -80 &#176;C. Furthermore, enzymatic assays of AWF should be performed as soon as possible after extraction to limit enzyme inactivation due to proteolysis, prolonged dilution or freeze-thawing.
The process of infiltration dilutes the apoplastic fluid and it is often necessary to determine the extent of this dilution. The centrifugation step may also dilute the AWF with water from intracellular compartments. A dilution factor is needed to estimate in vivo apoplast chemical concentrations when measurements are made on AWF. A dilution factor is also needed to concentrate AWF back to full strength when it is being used as an apoplast mimicking growth medium for microbes - thus matching in vivo metabolite concentrations as accurately as possible. Several variations exist on the method described in step 4 for determining the AWF dilution factor by measuring the dilution of a marker compound added to the infiltration fluid. For all methods the AWF dilution calculation assumes that the infiltration fluid is not appreciably absorbed or diluted by the leaf cells during the AWF recovery process. This assumption has been previously verified for the infiltration step14 but is unverified for the centrifugation step. The marker compound must also not be absorbed, transported or modified while in the apoplast. Indigo carmine is the most widely used and thoroughly tested of dyes used for AWF dilution calculations. Indigo carmine displays a low absorbance to cation exchange resin and isolated cell walls and was shown to be suitable for AWF calculations in Brassica napus, Pisum sativum, Solanum lycopersicum and Glycine max11,21,22. However, in some leaf types, e.g., rice and cucumber, indigo carmine appeared not be fully recovered after infiltration, which would lead to an underestimation of the AWF dilution factor12,21. The lack of recovery of indigo carmine may be due to its susceptibility to cleavage into isatin sulfonate by superoxide23, which is known to be produced in the apoplast, especially under stress24. Cleavage of indigo carmine into isatin sulfonate will result in a loss of absorbance at 610 nm and an increase at 245 nm. Whether this reaction occurs to an appreciable extent in the apoplast, or whether this reaction can be inhibited by the addition of superoxide scavengers to infiltration fluid has not been investigated to date. Blue dextran has been used instead of indigo carmine for quantification of the AWF dilution factor in rice leaves12, though its stability and recovery in AWF has not been reported. Alternatively, radiolabelled compounds such as [14C] sorbitol or other quantifiable internal standards can be used instead of dyes and the decrease in radioactivity or concentration measured and used to calculate the dilution factor in an analogous way11,14,25.
Limitations of the technique
A few caveats exist for the infiltration-centrifugation AWF isolation method. First, the dilution of the apoplast during infiltration may elicit a response from the plant. If the surrounding leaf cells detect a diminished concentration for certain components of the apoplastic fluid, they may respond by excreting further metabolites, distorting the interpretation of metabolite concentrations. In an assessment of this problem it was observed that neither the time between infiltration and centrifugation nor moderate differences in the ionic strength of the infiltration fluid affected the composition of the extracted AWF14,22. Therefore, any artifacts resulting from apoplast dilution are thought to be minimal.
A second potential drawback is that elution of AWF by centrifugation does not capture all the molecules present in the apoplast for several reasons. Some compounds, in particular cations and proteins may be tightly associated with the negatively charged cell wall and not elute with the AWF13. Other molecules such as proteins may be too large to elute efficiently out of the apoplast at the centrifugation speeds used14. Reactive oxygen species are an important class of compound produced in the apoplast, but due to the short-lived nature of these compounds, and unspecified requirements for their production, their presence is not well captured by AWF extraction. It is not known how accurately AWF represents the in vivo composition of the apoplast fluid and this may vary between species.
Several assays exist to determine cytoplasmic contamination, though none are universally accepted. Assays of predominantly cytoplasmic enzymes (e.g., G6PDH, hexose phosphate isomerase, MDH) have the benefit of being easy to perform but may not correlate well with cytoplasmic leakage14,18. The assessment of cytoplasmic metabolites (e.g., hexose phosphates, chlorophyll) is perhaps more indicative but is less well established11. Nevertheless, either type of assay can be useful for relative quantification of apoplastic contamination between samples. Ideally, multiple independent measurements should be used to validate the integrity of the AWF sample.
While acknowledging its limitations, the infiltration-centrifugation technique described here remains a simple and robust technique in the study of apoplastic proteins, primary and secondary metabolites and inorganic ions.

The plant apoplast is the intercellular space that surrounds plant cells. This is a dynamic environment in which many metabolic and transport processes take place. The major structural component of the apoplast is the cell wall, which is assembled and modified by enzymes, structural proteins and metabolites located within the apoplast. In healthy plant cells the apoplast is generally maintained in an acidic state allowing amino acids, sugars and other nutrients to be imported from the apoplast to the cytoplasm by H+ symport1. During sucrose transport, sucrose moves from photosynthetic sources through the apoplast and into phloem vasculature; sucrose is then transported by an osmotic potential maintained by apoplastic invertases cleaving sucrose in the sink organs2. Sugars and other nutrients can also be transported upwards and accumulate in stomatal cavities through the transpiration stream3.
The apoplast also represents an environmental niche where many pathogens establish their parasitic lifestyle. Bacterial plant pathogens have the ability to multiply to high densities in the apoplast, which they access through natural openings such as stomata or through wounds4. The concentration of amino acids and other nitrogen compounds in tomato apoplast have been shown to be sufficient to support the nutritional requirements of bacterial and fungal pathogens5,6. Primary defense responses towards microbial pathogens also occur in the apoplast, namely the production of reactive oxygen species by extracellular peroxidases and oxidases and the strengthening of the cell wall through cross-linking and callose deposition7. The plant cell wall is rich in secondary metabolites with anti-microbial activities; the biochemical nature of these secondary metabolites varies between species8.
As a result of the above-mentioned and other processes, leaf apoplastic fluid contains a variety of proteins, sugars, organic acids, amino acids, secondary metabolites, metals and other cations (e.g., Mg2+, K+, Na+, Ca2+, Fe2/3+). Solute concentrations in the apoplast of shoots are controlled largely by the balance of transport processes occurring between the apoplast and the xylem, phloem and cytoplasm9. However, metabolic reactions and microbial growth also consume or produce apoplastic solutes. The composition of the apoplast is known to differ between plant species and genotypes and in response to changing environmental conditions including light, nutrition and biotic and abiotic stresses9. By studying the composition of the apoplast and how it changes, including properties such as redox and osmotic potential, pH, nutrient/metabolite availability and enzymatic activities, one can gain novel insights into how plants respond to their environment. Understanding or characterizing the molecular changes that occur in the apoplast solution is complicated because it is a spatially structured and dynamic compartment in which metabolites and/or ions may be volatile, transient or associated with the cell wall and plasma membrane. Furthermore, different analytical techniques are required to cover the range of different chemical types.
To study the composition of the soluble apoplast, fluid usually needs to be extracted from the tissue. Several methods exist to extract apoplast fluid from various tissues, including a newly proposed filter strip method10, but the most established extraction method for leaves is infiltration-centrifugation. This technique has been evaluated previously11-13 and Lohaus et al. 200114 provide a thorough examination of many of the method&#8217;s technical parameters. As implied by the name, the infiltration-centrifugation technique is a two-step method that essentially involves replacement of the apoplastic air space with an aqueous infiltration fluid, which mixes with the native apoplastic fluid, followed by recovery of the infiltration/apoplastic fluid mixture by gentle centrifugation of the leaves. As the recovered fluid is diluted and does not contain all compounds present in the apoplast (see below), this fluid is commonly known as apoplast washing fluid (AWF), or sometimes intercellular washing fluid, rather than apoplastic fluid. The infiltration-centrifugation technique is easily scalable allowing single or pooled AWF samples of adequate volume to be generated and subjected to a wide range of downstream biochemical and analytical techniques (e.g., protein electrophoresis, enzyme activity measurement, NMR, many types of chromatography and mass spectrometry). Leaf AWF is also useful as an apoplast mimicking growth medium for studying the interaction of plant colonizing microbes with their environment 5.
In the following protocols we describe how to perform the infiltration-centrifugation technique using Phaseolus vulgaris cv. Tendergreen leaves, and provide some examples of downstream analyses with a focus on metabolomics. Importantly, methods to assess the quality of AWF are provided along with advice to optimize the procedure for different leaf types.

<table border="0" cellpadding="0" cellspacing="0" width="906">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Name of Material/ Equipment</td>
			<td style="width:291px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Eppendorf Microcentrifuge Tubes</td>
			<td style="width:291px;">Eppendorf</td>
			<td style="width:139px;">22364111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">razor blade</td>
			<td style="width:291px;">Fisher</td>
			<td>12-640&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">60 ml syringe</td>
			<td style="width:291px;">Becton Dickinson</td>
			<td style="width:139px;">300865</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">20 ml syringe</td>
			<td style="width:291px;">Becton Dickinson</td>
			<td style="width:139px;">300613</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">4 inch parafilm</td>
			<td style="width:291px;">Bemis</td>
			<td style="width:139px;">PM-996</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">side arm flask</td>
			<td>SciLabware</td>
			<td>12972831</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">vacuum source</td>
			<td style="width:291px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">5 ml pipette tips</td>
			<td style="width:291px;">Fisher</td>
			<td>50-813-28&#160;</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:309px;">centrifuge</td>
			<td style="width:291px;">Beckman Coulter</td>
			<td>392932</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Swinging bucket rotor</td>
			<td style="width:291px;">Beckman Coulter</td>
			<td>369702</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">indigo carmine</td>
			<td style="width:291px;">Sigma</td>
			<td style="width:139px;">I8130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">microplate reader</td>
			<td style="width:291px;">Tecan</td>
			<td style="width:139px;">Infinite 200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">96 well plates</td>
			<td style="width:291px;">Becton Dickinson</td>
			<td style="width:139px;">353072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">freeze dryer</td>
			<td style="width:291px;">SciQuip</td>
			<td style="width:139px;">Christ Alpha 2-4 LD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">microcentrifuge</td>
			<td style="width:291px;">biorad</td>
			<td>166-0612EDU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">oxaloacetic acid</td>
			<td style="width:291px;">Sigma</td>
			<td>O4126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">D-glucose-6-phosphate</td>
			<td style="width:291px;">Sigma</td>
			<td>G7250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">NADH</td>
			<td style="width:291px;">Roche</td>
			<td>10128023001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">MDH assay kit</td>
			<td style="width:291px;">Biovision</td>
			<td>K654-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">G6PDH assay kit</td>
			<td style="width:291px;">Sigma</td>
			<td>MAK015-1KT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">G-6-P assay kit</td>
			<td style="width:291px;">Biovision</td>
			<td>K657-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">ribitol</td>
			<td style="width:291px;">Sigma</td>
			<td style="width:139px;">A5502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">methanol</td>
			<td style="width:291px;">Sigma</td>
			<td style="width:139px;">650471</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">chloroform</td>
			<td style="width:291px;">Sigma</td>
			<td style="width:139px;">472476</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">vacuum concentrator</td>
			<td style="width:291px;">Thermor Scientific</td>
			<td>SC250EXP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">methoxyamine hydrochloride</td>
			<td style="width:291px;">Sigma</td>
			<td style="width:139px;">226904</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-Methyl-N-(trimethylsilyl) trifluoroacetamide</td>
			<td style="width:291px;">Sigma</td>
			<td style="width:139px;">394866</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Typical results for AWF extractions performed on healthy 3 week old P. vulgaris cv. Tendergreenleaves, using distilled water as the infiltration fluid are shown in Table 2. Young P. vulgaris leaves are amenable to infiltration, and at the centrifugation speed used here (1,000 x g) the removal of the majority of infiltration fluid by centrifugation can be seen directly as the leaves revert to their original green colour. P. vulgaris leaf fresh weight was on average 1.1 x g before infiltration and yielded 0.5 ml of AWF per gram fresh weight. The protein concentration of the AWF was determined to be 0.18 &#177; 0.08 mg/ml using a standard Bradford protein assay. The dilution factor for these leaves, calculated by measuring indigo carmine dilution, was 2.3 &#177; 0.3 fold. The above values can vary substantially between species and pilot experiments are required to determine the yield of AWF per gram leaf fresh weight, as well as the AWF protein concentration and dilution factor that can be expected for a given leaf type.
Damage to the integrity of the cells can occur both during the infiltration step, if the changes in pressure are too rapid, and during the centrifugation step if the centrifugal force is too high. It is therefore necessary to assay AWF samples for markers of cytoplasmic contamination; ideally, multiple independent assays are performed. Here, the results from three possible assays are reported in Table 2. In well-performed extractions of P. vulgaris AWF, G6PDH was undetectable by a standard enzyme assay. This is consistent with other reports where G6PDH activity becomes detectable only above a threshold centrifugal force12. In contrast, a baseline level of malate dehydrogenase activity (2.5 U/ml) was detectable in all P. vulgaris AWF samples using a standard metabolic assay. Increasing the centrifugal force above a threshold of 1,000 x g resulted in increased MDH activities (results not shown). As the apoplast from other species is known to contain endogenous MDH activity18, the amount detected here is considered genuine apoplastic activity and significant increases above this baseline level are indicative of contamination. Metabolites that are thought to be predominantly cytoplasmic, such as hexose-6-phosphates, can also be used to assess contamination of AWF. Here, GC-MS analysis detected zero or trace levels of G-6-P in AWF extractions. By contrast, in cut maize root sections, G-6-P was detected after AWF extraction at all centrifugation speeds and increased in concentration at higher speeds, indicating cytoplasmic contamination10.
Metabolite analysis by GC-MS of P. vulgaris leaf AWF typically yields 40 - 60 identifiable metabolites and an approximately equal number of unidentifiable compounds (Figure 2). Organic acids, simple sugars, and amino acids represent the bulk of the identifiable metabolites, however, secondary plant metabolites have also been detected and quantified from AWF11. Several example peaks from these different molecule classes are labeled in Figure 2. Further downstream analytical techniques are applicable to these AWF samples to quantify various molecules, for example: ICP-MS, NMR, HPLC, atomic absorption spectroscopy, protein mass spectrometry.
The protein component of the apoplast is also represented in the AWF as shown in the Coomassie Brilliant Blue stained SDS-PAGE gel in Figure 3. Among other roles, the apoplastic enzymes are responsible for the synthesis of the cell wall and the creation of extracellular reactive oxygen species. Proteomic studies of AWF from various species have identified dozens of individual proteins and shown that the protein component of the apoplast responds to environmental stress13,19. Ideally AWF extract should be free from contamination by cytoplasmic proteins such as Rubisco; however in practice this is difficult to achieve. The presence of a Rubisco protein band at ~53 kDa following SDS-PAGE provides a further qualitative assay for the integrity of AWF samples. For example, the sample loaded in lane 2 in Figure 3 contains a greater amount of Rubisco contamination that of lane 1.

<table border="0" cellpadding="0" cellspacing="0" class="jove_content" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Phaseolus vulgaris AWF</td>
		</tr>
		<tr>
			<td>Volume of AWF (ml g-1 leaf FW)</td>
			<td>0.49 &#177; 0.09&#160;</td>
		</tr>
		<tr>
			<td>Protein conc. (mg ml-1)</td>
			<td>0.18 &#177; 0.08&#160;</td>
		</tr>
		<tr>
			<td>Dilution factor</td>
			<td>2.3 &#177; 0.3</td>
		</tr>
		<tr>
			<td>G6PDH activity (U ml-1)</td>
			<td>none detected</td>
		</tr>
		<tr>
			<td>Glc-6-P (mg ml-1)</td>
			<td>none detected</td>
		</tr>
		<tr>
			<td>MDH activity (U ml-1)</td>
			<td>2.5 &#177; 0.9&#160;</td>
		</tr>
	</tbody>
</table>
Table 2. Typical results for AWF extractions from P. vulgaris cv. Tendergreen leaves.
<img alt="Figure 1" fo:content-width="6in" src="/files/ftp_upload/52113/52113fig1highres.jpg" /> 
Figure 1. Standard apoplast extraction workflow. The numbers refer to steps in the protocol.
<img alt="Figure 2" src="/files/ftp_upload/52113/52113fig2highres.jpg" /> 
Figure 2. An example GC-MS chromatogram of P. vulgaris cv. Tendergreen AWF.&#160; The numbered example metabolite peaks are: 1-malonate, 2-phosphate, 3-succinate, 4-unknown, 5-malate, 6-asparagine, 7-ribitol (internal standard), 8-citrate, 9-glucose, 10-inositol, 11-caffeic acid, 12-sucrose.
<img alt="Figure 3" fo:content-width="3in" src="/files/ftp_upload/52113/52113fig3highres.jpg" width="250" /> 
Figure 3. An example SDS-PAGE Coomassie stained gel of P. vulgaris AWF leaf extracts. This gel provides a comparison between the protein components of two AWF extractions that differ in the amount of cytoplasmic contamination by the abundant plastidial enzyme Rubisco. Following AWF extraction both samples were subjected to acetone protein precipitation in a 4 fold excess (v/v) of cold acetone and resolubilized in water at one tenth the original volume. Lanes 1 and 2 contain 40 &#181;g of protein. The band corresponding to the Rubisco large chain (~53 kDa) is indicated by an arrow. Mr: protein molecular weight markers.

Watch this Scientific Journal Video about The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example at JoVE.com

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 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 ...

the-infiltration-centrifugation-technique-for-extraction-apoplastic

Research

JoVE Journal

Biology

Testing the Physiological Barriers to Viral Transmission in Aphids Using Microinjection

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When an uninfected aphid feeds on an infected plant it contracts the virus through the plant phloem. Once ingested, the virus must pass from the insect gut to the hemolymph (the insect  blood ) and then must pass through the salivary gland, in order to be transmitted back to a new plant. An aphid may take up different viruses when munching on a plant, however only a small fraction will pass through the gut and salivary gland, the two main barriers for transmission to infect more plants. In the lab, we use physalis plants to study PLRV transmission. In this host, symptoms are characterized by stunting and interveinal chlorosis (yellowing of the leaves between the veins with the veins remaining green). The video that we present demonstrates a method for performing aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut is preventing viral transmission.
The video that we present demonstrates a method for performing Aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut or salivary gland is preventing viral transmission.

Aphids are effective transmitters of plant viruses. Aphid microinjection of virus, the procedure we will show you today, is a technique allowing researchers to inject virus directly into the hemocoel of the aphid, bypassing the gut, one of the 2 major barriers for virus transmission in a circulative manner. The same technique is also used to inject dsRNA for RNAi.

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When ...

Testing Nicotine Tolerance in Aphids Using an Artificial Diet Experiment

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well know to have insecticidal properties. One response of tobacco plants to herbivory, or being gnawed upon by insects, is to increase the production of this neurotoxic alkaloid. Here, we will demonstrate how to set up an experiment to address this question of whether a tobacco-adapted strain of the green peach aphid, Myzus persicae, can tolerate higher levels of nicotine than the a strain of this insect that does not infest tobacco in the field.

In this video and assay is demonstrated that tests the tolerance of nicotine to two types of aphids one that infests tobacco plants in the field and one that does not.

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well ...

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse 

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid peptide (A&#946;) and intraneuronal accumulation of hyperphosphorylated tau protein. Because cerebrospinal fluid (CSF) is in direct contact with the extracellular space of the brain, it provides a reflection of the biochemical changes in the brain in response to pathological processes. CSF from AD patients shows a decrease in the 42 amino-acid form of A&#946; (A&#946;42), and increases in total tau and hyperphosphorylated tau, though the mechanisms responsible for these changes are still not fully understood. Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses. Here, we demonstrate a refined cisterna magna puncture technique for CSF sampling from the mouse. This extremely gentle sampling technique allows serial CSF samples to be obtained from the same mouse at 2-3 month intervals which greatly minimizes the confounding effect of between-mouse variability in A&#946; or tau levels, making it possible to detect subtle alterations over time. In combination with A&#946; and tau ELISA, this technique will be useful for studies designed to investigate the relationship between the levels of CSF A&#946;42 and tau, and their metabolism in the brain in AD mouse models. Studies in Tg mice could provide important validation as to the potential of CSF A&#946; or tau levels to be used as biological markers for monitoring disease progression, and to monitor the effect of therapeutic interventions. As the mice can be sacrificed and the brains can be examined for biochemical or histological changes, the mechanisms underlying the CSF changes can be better assessed. These data are likely to be informative for interpretation of human AD CSF changes.

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse

Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses in human patients. Here, we demonstrate a refined cisterna magna puncture technique for serial CSF sampling from the mouse.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid ...

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is avoided when the herbivore is able to select other plants2. Antibiosis is the degree to which the plant affects the fitness of the herbivore feeding on it1.Tolerance is the degree to which the plant can withstand or repair damage caused by the herbivore, without compromising the herbivore's growth and reproduction1. The durability of herbivore resistance in an agricultural setting depends to a great extent on the resistance mechanism favored during crop breeding efforts3.
We demonstrate a no-choice experiment designed to estimate the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp. Several species of African grasses of the genus Brachiaria are valuable forage and pasture plants in the Neotropics, but they can be severely challenged by several native species of spittlebugs (Hemiptera: Cercopidae)4.To assess their resistance to spittlebugs, plants are vegetatively-propagated by stem cuttings and allowed to grow for approximately one month, allowing the growth of superficial roots on which spittlebugs can feed. At that point, each test plant is individually challenged with six spittlebug eggs near hatching. Infestations are allowed to progress for one month before evaluating plant damage and insect survival. Scoring plant damage provides an estimate of tolerance while scoring insect survival provides an estimate of antibiosis. This protocol has facilitated our plant breeding objective to enhance spittlebug resistance in commercial brachiariagrases5.

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

This video explains mechanisms of host plant resistance to herbivory and demonstrates a no-choice test that estimates the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp.

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is ...

Detection of Histone Modifications in Plant Leaves

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles1-2. H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues1-2. These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)3-7. 
Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde8,9, extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies9,10, de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C4 photosynthesis in maize5,11 and systemic immunity in Arabidopsis3.

A reliable and useful approach to detect histone modifications on specific plant genes is described. The approach combines chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. It allows detection of histone modifications on specific genes with a role in diverse physiological processes.

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent ...

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 ...

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2 When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3 Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5 by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.6-8

A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. These structures are almost always localized on plant surfaces. Among them are nectaries or glandular trichomes. The secreted compounds are often employed in interactions with the biotic environment, for example as attractants for pollinators or deterrents against herbivores.
Glands that are unique in several aspects can be found in carnivorous plants. In so-called pitcher plants of the genus Nepenthes, bifunctional glands inside the pitfall-trap on the one hand secrete the digestive fluid, including all enzymes necessary for prey digestion, and on the other hand take-up the released nutrients. Thus, these glands represent an ideal, specialized tissue predestinated to study the underlying molecular, biochemical, and physiological mechanisms of protein secretion and nutrient uptake in plants. Moreover, generally the biosynthesis of secondary compounds produced by many plants equipped with glandular structures could be investigated directly in glands.
In order to work on such specialized structures, they need to be isolated efficiently, fast, metabolically active, and without contamination with other tissues. Therefore, a mechanical micropreparation technique was developed and applied for studies on Nepenthes digestion fluid. Here, a protocol is presented that was used to successfully prepare single bifunctional glands from Nepenthes traps, based on a mechanized microsampling platform. The glands could be isolated and directly used further for gene expression analysis by PCR techniques after preparation of RNA.

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Plants glands are specialized structures responsible for the biosynthesis and secretion of many compounds involved in interactions with the biotic environment. To enable studies on their molecular and biochemical features, a mechanical micropreparation technique was established in order to isolate single metabolically active glands, here from the carnivorous plant Nepenthes.

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used as manufacturing platforms for added-value products such as biopharmaceuticals. In both cases, liquid plant extracts may need to be clarified to remove particulates. Optimal clarification reduces the costs of filtration and centrifugation by increasing capacity and longevity. This can be achieved by introducing charged polymers known as flocculants, which cross-link dispersed particles to facilitate solid-liquid separation. There are no mechanistic flocculation models for complex mixtures such as plant extracts so empirical models are used instead. Here a design-of-experiments procedure is described that allows the rapid screening of different flocculants, optimizing the clarification of plant extracts and significantly reducing turbidity. The resulting predictive models allow the identification of robust process conditions and sets of polymers with complementary properties, e.g. effective flocculation in extracts with specific conductivities. The results presented for tobacco leaf extracts can easily be adapted to other plant species or tissues and will thus facilitate the development of more cost-effective downstream processes for commodities and plant-derived pharmaceuticals.

The design-of-experiments procedure presented here allows the evaluation of different flocculants in terms of their ability to aggregate dispersed particles in plant extracts, thus reducing turbidity and the costs of downstream processing.

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...