We thank Jennifer Deke and Urszula Zarzenska for their scientific support. This work was financially supported by a Career Integration Grant (CIG, PCIG14-GA-2013-63 0734) by the European Commission within the 7th framework program, the grant LFF-GK06 &#39;DELIGRAH&#39; (Landesforschungsf&#246;rderung Hamburg), and the DFG grant (DFG KE 856_6-1) awarded to JK.

1. Sampling and preparation of phloem sap from B. napus plants 4,8,20
<ol>
	<li>For phloem sap sampling, use well-watered 8-week-old B. napus plants that show only initial flowering to prevent pollen contaminations.</li>
	<li>Use a hypodermic needle (&#216; 0.8 mm) and punctate the inflorescence stem several times. Repeat at several other inflorescence stems and on other plants to obtain enough phloem sap (Figure 2a). 
	NOTE: For complex analysis, it is recommended to start with at least 600 &#181;L of phloem sap. 4 to 5 plants will yield between 200 and 700 &#181;L of phloem sap.</li>
	<li>Wipe off the first drop from the injured sites with filter paper. These drops mainly contain highly contaminated material from surrounding injured tissue and need to be discarded.</li>
	<li>Collect the following drops by pipetting and store the collected exudate in a pre-chilled conical 1.5 mL reaction tube at -20 &#176;C using an ice-free cooling system.</li>
	<li>Continue collecting until no further drop formation is observable, but no longer than 1 h. This procedure can be repeated after two days without significant loss of collected phloem sap. 
	NOTE: The collected phloem sap can be used immediately or stored at -80 &#176;C for future analysis. For -80 &#176;C storage, shock-freeze the reaction tube in liquid nitrogen.</li>
	<li>Concentrate the phloem sample to approximately 1/6th of the initial volume using centrifugal concentrators with a molecular weight cut off (MWCO) of 10,000 Daltons (Da). Centrifuge the concentrated sample at 4 &#176;C and 20,000 x g for at least 15 min to remove dust particles. 
	NOTE: Phloem sap concentration does not lead to a significant loss of proteins.</li>
	<li>For subsequent complex analysis, proceed with step 3.1.</li>
</ol>
2. Phloem sap purity control using reverse transcriptase PCR (RT-PCR) 4,8,20
<ol>
	<li>Isolate total RNA from phloem sap using a standard RNA extraction method for liquid material (e.g. TRIzol or phenol-chloroform extraction followed by isopropanol precipitation from the aqueous phase and ethanol washing steps according to the manufacturer&#39;s protocol). 
	Caution: Phenol-chloroform is toxic. Work under a hood with appropriate personal protection equipment. The extracted RNA can be stored in ethanol at -80 &#176;C for up to six months.</li>
	<li>Remove contamination of DNA by digestion with DNase I (1 u/&#181;g) for 45 min at 37 &#176;C and inactivate the enzyme by adding EDTA to a final concentration of 5 mM and incubate the sample at 75 &#176;C for 10 min.</li>
	<li>To get pure RNA, purify and concentrate the sample by another purification step (e.g. using RNA Cleanup Kits, according to the manufacturer&#39;s instructions).</li>
	<li>Perform a reverse transcriptase PCR (RT-PCR) for synthesis of cDNA with a cDNA synthesis kit using 150 ng of pure RNA as described by the manufacturer&#39;s protocol. 
	NOTE: The cDNA can be stored at -20 &#176;C until further use.</li>
	<li>Use 5 &#181;L of cDNA as a template for a standard PCR reaction as specified by the manufacturer, to amplify compartment-specific transcripts and check by agarose gel electrophoresis. Confirm the purity of phloem samples by e.g. using primers for rubisco small subunit, thioredoxin h, and the pollen coat protein (Figure 1b, Table 1).</li>
</ol>
3. Blue native PAGE 4,25,26,31
<ol>
	<li>Use either self-poured native gradient gels as described elsewhere27 or commercial Bis-Tris gradient gels.</li>
	<li>Load 20 to 30 &#181;g of the concentrated protein sample per well onto the gel in at least triplicates.</li>
	<li>Run the gel at 4 &#176;C and 150 V using dark blue cathode buffer B and anode buffer (Table 2) until the sample passed 1/3rd of the gel (ca. 20 to 30 min, Figure 3a). 
	NOTE: For northern blotting, a single gel lane is sufficient, while for the analysis of protein composition by 3D PAGE and mass spectrometry it is recommended to combine the same band from up to ten gel lanes to concentrate less abundant proteins.</li>
	<li>Pause the run and exchange dark blue cathode buffer B with light blue cathode buffer B/10 (Table 2) and continue the run. Finish the gel run when the blue front starts to elute out of the gel (ca. 120 to 180 min, Figure 3a). 
	NOTE: The finished blue native gel can be stored at 4 &#176;C for at least one week. Prolonged storage can lead to diffuse bands and should be avoided. Dark blue buffer B can be reused.</li>
</ol>
4. OPTIONAL: Detection of RNA using blue native northern blotting 4
<ol>
	<li>Cut out distinct bands or use the whole gel lane from the Blue native gel and transfer them onto a nylon membrane by semi-dry blotting at 3.5 mA/cm2 for 60 min and mark the upper and lower gel border with a pencil on the membrane (Figure 4a).</li>
	<li>After blotting, wash the membrane with DEPC-treated water and dry between two filter papers.</li>
	<li>Perform UV-crosslinking of the blotted membrane with 120,000 &#181;J/cm2 (85 s if using the Crosslinker in Table of Materials).</li>
	<li>Pre-hybridize the membrane with 6 mL of hybridization buffer (commercial or self-made) for 60 min at 68 &#176;C in a hybridization oven (Figure 4a).</li>
	<li>Add an additional 1 mL of hybridization buffer containing 4 &#181;L of 3&#39;-biotinylated probes (100 nM).</li>
	<li>Incubate the membrane with the probe overnight, while cooling the oven from 68 &#176;C to 37 &#176;C.</li>
	<li>Wash the membrane with buffer containing 2x SSC and 0.1 % SDS, for 5 min at room temperature to remove probes bound unspecifically. 
	Caution: SDS can cause irritations. Wear gloves and lab coat when working with SDS and SDS containing solutions.</li>
	<li>Perform the detection of the 3&#39;-biotinylated probes on the membrane e.g. with a Streptavidin-HRP-conjugate and luminol as a horseradish peroxidase (HRP) substrate, resulting in a sensitive chemiluminescent reaction (Figure 4a).</li>
</ol>
5. Second dimension: Tris-Tricine-urea PAGE 4,28
<ol>
	<li>Excise single bands from the blue native gel. Combine bands from samples run in parallel lanes to achieve a further concentration of complex proteins (Figure 5). 
	NOTE: Excised bands can be stored in single reaction tubes until further use at 4 &#176;C.</li>
	<li>Pour a 12 % Tris-Tricine-urea gel (thickness of 1 mm, 6.8cm x 8.6 cm (width x length)). Prepare 10 mL of gel solution A for the separating gel (Table 2), pour between two glass plates and overlay with isopropanol. Remove the isopropanol after the polymerization is finished and transfer 4 mL of gel solution B (Table 2) for the stacking gel onto the gel and insert a 10 well comb. 
	Caution: Unpolymerized acrylamide can be neurotoxic and TEMED is harmful if inhaled. Always wear personal protection equipment and work under a hood.</li>
	<li>Transfer the excised gel bands into a 1.5 mL reaction tube and add 1 mL of 2x SDS sample buffer for equilibration of the gel pieces (Figure 5, Table 2).
	<ol>
		<li>Incubate the samples for 10 min at room temperature, prior to boiling in a heating block (95 &#176;C) or in a microwave and incubate the samples again at room temperature for a further 15 min.</li>
		<li>Stack several equilibrated gel pieces representing the same complex into one single gel pocket.</li>
		<li>Perform the electrophoresis using anode and cathode running buffers at 150 V for 45 to 60 min and excise the whole gel lane.</li>
	</ol>
	</li>
	<li>Incubate the cut lane for 20 min in acidic buffer (100 mM Tris, 100 mM acetic acid) and equilibrate in 125 mM Tris-HCl, pH 6.8 for another 20 min (Figure 5).</li>
</ol>
6. Third dimension: SDS-PAGE 4,32
<ol>
	<li>Pour a 15 % SDS separating gel according to Laemmli32 (thickness: 1.5 mm, 6.8cm x 8.6 cm (Width x Length)) (Table 2) and add a thin layer of a 4 % stacking gel above. 
	Caution: SDS, acrylamide and TEMED are toxic and harmful. Work under a hood and wear personal protection equipment.</li>
	<li>Transfer the equilibrated gel lane onto the SDS gel and cover the gel with 0.5 % (w/w) agarose in 1x SDS running buffer supplemented with a trace of bromophenol blue (Figure 5).</li>
	<li>Boil the SDS running buffer with agarose in the microwave prior loading onto the gel.</li>
	<li>For running a protein marker to estimate the molecular weights of the single components, insert a piece of filter paper on one end of the gel until the agarose solidified or use a special comb typically used for IPG gels.</li>
	<li>Run the gel at 150 V for 60 min and stain the gel with either colloidal Coomassie, Silver stain or any other staining method suitable for subsequent mass spectrometric analysis.</li>
	<li>Analyze the visible protein spots using mass spectrometric approaches like matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) or LC-MS/MS mass spectrometry. 
	NOTE: We recommend to use the colloidal Coomassie staining as already described33. In our hands, even less abundant proteins can be sufficiently stained and allows a mass spectrometric analysis with reasonable data quality.</li>
</ol>

<ol>
	<li>Kempers, R., van Bel, A. J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symplasmic+connections+between+sieve+element+and+companion+cell+in+the+stem+phloem+ofVicia+faba+L.+have+a+molecular+exclusion+limit+of+at+least+10+kDa.">Symplasmic connections between sieve element and companion cell in the stem phloem ofVicia faba L. have a molecular exclusion limit of at least 10 kDa.</a> Planta. 201 (2), 195-201 (1997).</li><li>Stadler, R., 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=Expression+of+GFP-fusions+in+Arabidopsis+companion+cells+reveals+non-specific+protein+trafficking+into+sieve+elements+and+identifies+a+novel+post-phloem+domain+in+roots.">Expression of GFP-fusions in Arabidopsis companion cells reveals non-specific protein trafficking into sieve elements and identifies a novel post-phloem domain in roots.</a> Plant J. 41 (2), 319-331 (2005).</li><li>Imlau, A., Truernit, E., Sauer, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-to-cell+and+long-distance+trafficking+of+the+green+fluorescent+protein+in+the+phloem+and+symplastic+unloading+of+the+protein+into+sink+tissues.">Cell-to-cell and long-distance trafficking of the green fluorescent protein in the phloem and symplastic unloading of the protein into sink tissues.</a> Plant Cell. 11 (3), 309-322 (1999).</li><li>Ostendorp, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+Brassica+napus+phloem+protein+and+ribonucleoprotein+complexes.">Functional analysis of Brassica napus phloem protein and ribonucleoprotein complexes.</a> New Phytol. 214 (3), 1188-1197 (2017).</li><li>Paultre, D. S. G., Gustin, M. -. P., Molnar, A., Oparka, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lost+in+Transit:+Long-Distance+Trafficking+and+Phloem+Unloading+of+Protein+Signals+in+Arabidopsis+Homografts.">Lost in Transit: Long-Distance Trafficking and Phloem Unloading of Protein Signals in Arabidopsis Homografts.</a> Plant Cell. 28 (9), 2016-2025 (2016).</li><li>Sali, A., Glaeser, R., Earnest, T., Baumeister, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+words+to+literature+in+structural+proteomics.">From words to literature in structural proteomics.</a> Nature. 422 (6928), 216-225 (2003).</li><li>Lin, M. -. K., Lee, Y. -. J., Lough, T. J., Phinney, B. S., Lucas, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+Pumpkin+Phloem+Proteome+Provides+Insights+into+Angiosperm+Sieve+Tube+Function.">Analysis of the Pumpkin Phloem Proteome Provides Insights into Angiosperm Sieve Tube Function.</a> Mol. Cell. Proteomics. 8 (2), 343-356 (2008).</li><li>Giavalisco, P., Kapitza, K., Kolasa, A., Buhtz, A., Kehr, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+the+proteome+of+Brassica+napus+phloem+sap.">Towards the proteome of Brassica napus phloem sap.</a> Proteomics. 6 (3), 896-909 (2006).</li><li>Lucas, W. J., Yoo, B. C., Kragler, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+as+a+long-distance+information+macromolecule+in+plants.">RNA as a long-distance information macromolecule in plants.</a> Nat. Rev. Mol. Cell. Bio. 2 (11), 849-857 (2001).</li><li>Dinant, S., Kehr, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sampling+and+Analysis+of+Phloem+Sap.">Sampling and Analysis of Phloem Sap.</a> Plant Mineral Nutrients. 953, 185-194 (2013).</li><li>Kennedy, J. S., Mittler, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Method+of+obtaining+Phloem+Sap+via+the+Mouth-parts+of+Aphids.">A Method of obtaining Phloem Sap via the Mouth-parts of Aphids.</a> Nature. 171 (4351), 528 (1953).</li><li>King, R. W., Zeevaart, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+Phloem+exudation+from+cut+petioles+by+chelating+agents.">Enhancement of Phloem exudation from cut petioles by chelating agents.</a> Plant Physiol. 53 (1), 96-103 (1974).</li><li>Alosi, M. C., Melroy, D. L., Park, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+of+gelation+of+Phloem+exudate+from+cucurbita+fruit+by+dilution,+glutathione,+and+glutathione+reductase.">The regulation of gelation of Phloem exudate from cucurbita fruit by dilution, glutathione, and glutathione reductase.</a> Plant Physiol. 86 (4), 1089-1094 (1988).</li><li>Dinant, S., Bonnemain, J. -. L., Girousse, C., Kehr, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phloem+sap+intricacy+and+interplay+with+aphid+feeding.">Phloem sap intricacy and interplay with aphid feeding.</a> Comptes rendus biologies. 333 (6-7), 504-515 (2010).</li><li>Tetyuk, O., Benning, U. F., Hoffmann-Benning, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collection+and+analysis+of+Arabidopsis+phloem+exudates+using+the+EDTA-facilitated+Method.">Collection and analysis of Arabidopsis phloem exudates using the EDTA-facilitated Method.</a> J Vis Exp. (80), e51111 (2013).</li><li>Kovalskaya, N., Owens, R., Baker, C. J., Deahl, K., Hammond, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+modified+EDTA-mediated+exudation+technique+and+guttation+fluid+analysis+for+Potato+spindle+tuber+viroid+RNA+detection+in+tomato+plants+(Solanum+lycopersicum).">Application of a modified EDTA-mediated exudation technique and guttation fluid analysis for Potato spindle tuber viroid RNA detection in tomato plants (Solanum lycopersicum).</a> J. Virol. Methods. 198, 75-81 (2014).</li><li>Rodriguez-Medina, C., Atkins, C. A., Mann, A. J., Jordan, M. E., Smith, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecular+composition+of+phloem+exudate+from+white+lupin+(Lupinus+albus+L).">Macromolecular composition of phloem exudate from white lupin (Lupinus albus L).</a> BMC Plant Biol. 11 (1), 36 (2011).</li><li>Taylor, J. S., Thompson, B., Pate, J. S., Atkins, C. A., Pharis, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokinins+in+the+Phloem+Sap+of+White+Lupin+(Lupinus+albus+L.).">Cytokinins in the Phloem Sap of White Lupin (Lupinus albus L.).</a> Plant Physiol. 94 (4), 1714-1720 (1990).</li><li>Yoo, B., 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=A+Systemic+Small+RNA+Signaling+System+in+Plants.">A Systemic Small RNA Signaling System in Plants.</a> Plant Cell. 16 (8), 1979-2000 (2004).</li><li>Buhtz, A., Springer, F., Chappell, L., Baulcombe, D. C., Kehr, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+small+RNAs+from+the+phloem+of+Brassica+napus.">Identification and characterization of small RNAs from the phloem of Brassica napus.</a> Plant J. 53 (5), 739-749 (2008).</li><li>Chalhoub, B., 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=Plant+genetics.+Early+allopolyploid+evolution+in+the+post-Neolithic+Brassica+napus+oilseed+genome.">Plant genetics. Early allopolyploid evolution in the post-Neolithic Brassica napus oilseed genome.</a> Science. 345 (6199), 950-953 (2014).</li><li>Sasaki, T., Chino, M., Hayashi, H., Fujiwara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+several+mRNA+species+in+rice+phloem+sap.">Detection of several mRNA species in rice phloem sap.</a> Plant Cell Phys. 39 (8), 895-897 (1998).</li><li>Doering-Saad, C., Newbury, H. J., Bale, J. S., Pritchard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+aphid+stylectomy+and+RT-PCR+for+the+detection+of+transporter+mRNAs+in+sieve+elements.">Use of aphid stylectomy and RT-PCR for the detection of transporter mRNAs in sieve elements.</a> J Exp. Bot. 53 (369), 631-637 (2002).</li><li>Ruiz-Medrano, R., Xoconostle-C&#225;zares, B., Lucas, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phloem+long-distance+transport+of+CmNACP+mRNA:+implications+for+supracellular+regulation+in+plants.">Phloem long-distance transport of CmNACP mRNA: implications for supracellular regulation in plants.</a> Dev. 126 (20), 4405-4419 (1999).</li><li>Sch&#228;gger, H., Cramer, W. A., von Jagow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+molecular+masses+and+oligomeric+states+of+protein+complexes+by+blue+native+electrophoresis+and+isolation+of+membrane+protein+complexes+by+two-dimensional+native+electrophoresis.">Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis.</a> Anal. Biochem. 217 (2), 220-230 (1994).</li><li>Sch&#228;gger, H., von Jagow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+native+electrophoresis+for+isolation+of+membrane+protein+complexes+in+enzymatically+active+form.">Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form.</a> Anal. Biochem. 199 (2), 223-231 (1991).</li><li>Fiala, G. J., Schamel, W. W. A., Blumenthal, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+native+polyacrylamide+gel+electrophoresis+(BN-PAGE)+for+analysis+of+multiprotein+complexes+from+cellular+lysates.">Blue native polyacrylamide gel electrophoresis (BN-PAGE) for analysis of multiprotein complexes from cellular lysates.</a> J Vis Exp. (48), e2164 (2011).</li><li>Rais, I., Karas, M., Sch&#228;gger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+electrophoresis+for+the+isolation+of+integral+membrane+proteins+and+mass+spectrometric+identification.">Two-dimensional electrophoresis for the isolation of integral membrane proteins and mass spectrometric identification.</a> Proteomics. 4 (9), 2567-2571 (2004).</li><li>Sch&#228;gger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tricine-SDS-PAGE.">Tricine-SDS-PAGE.</a> Nature protocols. 1 (1), 16-22 (2006).</li><li>Wittig, I., Braun, H. -. P., Sch&#228;gger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+native+PAGE.">Blue native PAGE.</a> Nat Protoc. 1 (1), 418-428 (2006).</li><li>Eubel, H., Braun, H. -. P., Millar, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue-native+PAGE+in+plants:+a+tool+in+analysis+of+protein-protein+interactions.">Blue-native PAGE in plants: a tool in analysis of protein-protein interactions.</a> Plant Methods. 1 (1), 11 (2005).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227 (5259), 680-685 (1970).</li><li>Dyballa, N., Metzger, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+sensitive+colloidal+coomassie+G-250+staining+for+proteins+in+polyacrylamide+gels.">Fast and sensitive colloidal coomassie G-250 staining for proteins in polyacrylamide gels.</a> J Vis Exp. (30), (2009).</li><li>Turnbull, C. G. N., Lopez-Cobollo, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heavy+traffic+in+the+fast+lane:+long-distance+signalling+by+macromolecules.">Heavy traffic in the fast lane: long-distance signalling by macromolecules.</a> New Phytol. 198 (1), 33-51 (2013).</li><li>Hall, S. M., Baker, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemical+composition+of+Ricinus+phloem+exudate.">The chemical composition of Ricinus phloem exudate.</a> Planta. 106 (2), 131-140 (1972).</li><li>Barnes, A., Bale, J., Constantinidou, C., Ashton, P., Jones, A., Pritchard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+protein+identity+from+sieve+element+sap+in+Ricinus+communis+L.+by+quadrupole+time+of+flight+(Q-TOF)+mass+spectrometry.">Determining protein identity from sieve element sap in Ricinus communis L. by quadrupole time of flight (Q-TOF) mass spectrometry.</a> J. Exp. Bot. 55 (402), 1473-1481 (2004).</li><li>Walz, C., Giavalisco, P., Schad, M., Juenger, M., Klose, J., Kehr, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+of+curcurbit+phloem+exudate+reveals+a+network+of+defence+proteins.">Proteomics of curcurbit phloem exudate reveals a network of defence proteins.</a> Phytochemistry. 65 (12), 1795-1804 (2004).</li><li>Walz, C., Juenger, M., Schad, M., Kehr, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+the+presence+and+activity+of+a+complete+antioxidant+defence+system+in+mature+sieve+tubes.">Evidence for the presence and activity of a complete antioxidant defence system in mature sieve tubes.</a> Plant J. 31 (2), 189-197 (2002).</li><li>Kehr, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phloem+sap+proteins:+their+identities+and+potential+roles+in+the+interaction+between+plants+and+phloem-feeding+insects.">Phloem sap proteins: their identities and potential roles in the interaction between plants and phloem-feeding insects.</a> J Exp. Bot. 57 (4), 767-774 (2006).</li><li>Ham, B. -. K., Brandom, J. L., Xoconostle-C&#225;zares, B., Ringgold, V., Lough, T. J., Lucas, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+polypyrimidine+tract+binding+protein,+pumpkin+RBP50,+forms+the+basis+of+a+phloem-mobile+ribonucleoprotein+complex.">A polypyrimidine tract binding protein, pumpkin RBP50, forms the basis of a phloem-mobile ribonucleoprotein complex.</a> Plant Cell. 21 (1), 197-215 (2009).</li><li>Schamel, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotinylation+of+protein+complexes+may+lead+to+aggregation+as+well+as+to+loss+of+subunits+as+revealed+by+Blue+Native+PAGE.">Biotinylation of protein complexes may lead to aggregation as well as to loss of subunits as revealed by Blue Native PAGE.</a> J. Immunol. Methods. 252 (1-2), 171-174 (2001).</li><li>Zhang, S., Sun, L., Kragler, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phloem-delivered+RNA+pool+contains+small+noncoding+RNAs+and+interferes+with+translation.">The phloem-delivered RNA pool contains small noncoding RNAs and interferes with translation.</a> Plant Physiol. 150 (1), 378-387 (2009).</li></ol>

The authors have nothing to disclose.

The phloem is a compartment of high interest, since it constitutes the major transport route for photoassimilates, and is also essential for long-distance signaling between different plant parts9,20,34,35. In addition to small molecules, macromolecules like proteins and RNAs have been implicated with phloem maintenance and signaling4,7,8. The analysis of phloem composition is restricted by the limited accessibility to phloem samples in most plant species. The insect stylet technique is widely applicable, but experimentally demanding and results in very small amounts of phloem samples11. One easy technique used for phloem sampling is EDTA-facilitated exudation12. EDTA chelates divalent ions like Calcium and thus prevents closing of sieve plates by P-proteins and callose. It is easy to use, but is prone to contamination by damaged cells and samples are diluted. Depleting divalent ions by EDTA could also lead to a breakdown of existing complexes. However, it allows sampling from a wide range of species including the model plant A. thaliana15. Spontaneous exudation of phloem sap only occurs in a small number of plant species. It is an invasive method that involves significant injury of plant tissues10. We recently established B. napus as a model species for studying long-distance transport4,8,20. Here, phloem sap can be sampled from small incisions, which reduces wounding effects and sources of contamination.
Using different sampling techniques, the proteome of phloem samples from different plant species has been investigated in recent years7,8,17,36,37,38,39. For these proteome studies, proteins were extracted and precipitated under denaturing conditions and therefore do not allow conclusions about protein:protein and protein:nucleic acid interactions present under native conditions. Co-immunoprecipitation (CoIP) and overlay assays indicated that a major ribonucleoprotein complex might exist in phloem sap from pumpkin40, but it was not demonstrated that any macromolecular complexes exist under native conditions within the phloem system.
Blue native PAGE, introduced by Sch&#228;gger et al.26, in combination with subsequent high-resolution gel electrophoresis steps enable the separation of the individual components of large protein complexes. Using 3D-PAGE analyses of native B. napus phloem exudate, we could recently demonstrate that several high-molecular-weight protein:protein and RNP complexes exist in phloem samples and are enzymatically active4. Alternative methods to isolate protein complexes as well as RNPs like size exclusion chromatography might exist, but fail in terms of resolution when compared to BN-PAGE. Furthermore, for a reasonable quality of complex separation, size exclusion column matrices have to be adapted according to the overall complex molecular weights being investigated. Analyzing complexes of different size will therefore demand different types of columns which is cost intensive and does not allow as high focusing power as a BN-PAGE.
Critical steps to analyze complexes from B. napus include the total amount of phloem sap used as starting material. At least 600 &#181;L of phloem sample are necessary and can be obtained from five well-watered plants. For 3D-PAGE and BN northern blotting it is necessary to start the initial BN gel with a sufficient amount of phloem samples. To achieve this, phloem samples are concentrated until 20 to 30 &#181;g of protein can be loaded into each well and at least triplicates of the same sample are run in parallel. Too low or too high concentrations reduce the detection of complexes (Figure 3). Phloem samples need be collected into reaction tubes on ice and should be stored frozen for later use to avoid protein degradation and turnover. Protease inhibitors have been found in all phloem proteomes analyzed, but also a fully active proteasome was described in the phloem of B. napus4. Furthermore, volume and composition of phloem samples depend on the sampling time-point and environmental conditions10. Therefore care must be taken to collect at the same time of the day under similar conditions. Stress treatments (e.g. drought) can reduce the amount of phloem that can be collected. To identify single proteins by mass spectrometry, it is mandatory to limit possible keratin contaminations by wearing gloves all the time. Keratin leads to additional signals during mass spec analysis and hampers protein identification. Running the BN-PAGE at a higher voltage or at ambient temperature can lead to heating of the gel that might result in disassembly of fragile complexes.
The protocol presented here is suitable for the analysis of protein:protein complexes. We also show that it can be used to detect specific RNAs contained in the complexes in parallel. To achieve this, we recently introduced a protocol for BN northern blotting4. RNAs are prone to degradation by RNAse contaminations. To limit such degradation DEPC-treated, water should be used.
Main limitations of the presented method include the estimation of the molecular weight of protein complexes separated by BN-PAGE, since migration behavior depends on size, shape and even on posttranslational modifications of the complexes31,41. Size estimation of RNP complexes is even more complicated due to the influence of nucleic acids on the migration behavior. It can also not be prevented that complexes of the same size co-migrate in one single band. This can lead to the erroneous prediction of complex compositions. Therefore verifying the observed interactions with complementary techniques, e.g. by functional assays4, might be necessary. Also proteins only weakly or transiently associated to a macromolecular complex might be lost in the BN buffer used. To avoid this, samples could be crosslinked, what can also lead to artifacts or can prevent protein identification, or the buffer conditions can be optimized.
In contrast to classical 2D-PAGE, the combination of urea- and SDS-PAGE allows the separation according to hydrophobicity and molecular weight instead of isoelectric point (pI) and molecular weight 28, and does not limit the separation to proteins of a specific pI range. Similar to classical 2D-PAGE, separated proteins are visible as single spots, but on a diagonal line 4,28 (Figure 5, Figure 6). More hydrophobic proteins appear in spots above this diagonal, whereas more hydrophilic proteins are found below the line28. The polyacrylamide concentrations used in this protocol will separate proteins between 25 to 80 kDa well. To allow a separation of smaller or larger proteins the polyacrylamide concentration can be varied or gradient gels can be used in the third dimension. The components of complex IV (Figure 3) separated by 3D-PAGE (Figure 6) were cut out, trypsin digested, and analyzed by mass spectrometry. This resulted in the identification of several tRNA ligases. The components of the other complexes have been published recently 4. Our 3D-PAGE enabled the separation of different ligases, namely aspartate, asparagine, threonine, lysine and glycine ligases, with similar molecular weights (Table 3). Using a methionine tRNA-specific probe, we were able to detect potentially bound tRNA within complex IV, but if full-length tRNA or tRNA fragments are in the complex cannot be discriminated with the probes used. To check if the nucleic acids are really bound within the complex and not co-migrating, protease digested phloem sap samples might serve as suitable migration controls.
It has been speculated earlier that protein translation can occur within phloem sieve tubes, since almost most components of the entire ribosome as well as elongation factors have been found in phloem samples4,7. Our finding of tRNA and tRNA ligases seems to support this idea. However, tRNA fragments present in phloem samples from pumpkin have been shown to be potent inhibitors of translation42 and functional ribosomes seem to be absent4, suggesting that translation cannot take place.
Taken together, the protocol presented here allows the separation of native protein:protein and even RNP complexes from phloem samples and the separation and identification of their individual components with high resolution. The application of this method enables the discovery of novel protein and RNP complexes in phloem samples and can therefore elucidate new functions in this highly specialized plant compartment.

The long-distance conduits of the phloem are essential for the allocation of organic nutrients in higher plants, but are also involved in regulating many different processes important for growth and development, pathogen defense, and the adaptation to adverse environmental conditions. Besides small effectors like ions, phytohormones, or metabolites themselves, macromolecules like proteins and RNAs have recently emerged as potential signals.
Studies have shown that phloem loading of proteins is size dependent and controlled by the size exclusion limit (SEL) of the pore-plasmodesmal units connecting companion cells (CC) and sieve elements (SE) that are typically in the range of 10 to &#62;67 kDa1,2,3. However, despite these size limitations larger proteins and protein complexes have been found within the phloem system challenging the idea of size exclusion4, and even nonspecific loss of proteins from CC to SE has been suggested5.
Multiprotein as well as large ribonucleoprotein complexes play pivotal roles in biosynthesis and maintaining the structural integrity of the cell6. There is evidence that phloem-mobile protein-protein and ribonucleoprotein complexes exist4,7, but to date little about their function is known. In phloem sieve elements protein:protein and RNP complexes have been implicated with long-distance transport and/ or signaling8,9. To unravel the functions of the translocated complexes, studying their composition under varying environmental or stress conditions is required. To achieve this, native complexes have to be isolated and their components have to be separated with sufficient resolution.
To isolate high-molecular-weight complexes from the phloem, it is first necessary to obtain sap samples in sufficient quality and quantity. The technique that can be used for phloem sampling depends on the plant species of interest. Three main methods to obtain phloem sap exist 10: insect stylectomy11, EDTA-facilitated exudation12, and spontaneous exudation13.
Phloem samples from Arabidopsis thaliana (A. thaliana), the major model plant in laboratories worldwide, can only be obtained by EDTA-facilitated exudation or aphid stylectomy14,15. Both methods lead to either highly diluted phloem sap or very low sample amounts. EDTA-facilitated exudation is also prone to contamination and might not represent the real phloem composition16. Spontaneous phloem exudation is limited to plant species like cucurbits, lupine or yucca, where cutting off plant parts leads to a spontaneous emission of phloem sap that can be easily collected13,17,18,19. We recently established oilseed rape (Brassica napus) as a suitable model system for phloem analysis, since it is a close relative of A. thaliana and several microliters of phloem sap can be obtained from small incisions that has been shown to be of high purity4,8,20. Furthermore, the B. napus genome sequence was released in 201421.
Except for aphid stylectomy, all phloem collection methods described include damaging of surrounding tissue at the site of sampling, and the composition of phloem sap may change in response to injury. Therefore it is essential to check the quality of phloem samples, regardless of the sampling method applied. There are different methods for quality control of collected phloem sap, e.g. by determination of RNase activity22,23, RT-PCR with primers against Rubisco8,24, or the analysis of the sugar composition8.
Different methods to isolate and separate protein complexes can be applied, including size exclusion chromatography, sucrose density ultracentrifugation, or sample filtration through membranes with distinct molecular weight cut offs. However, these approaches do not allow high resolution. The technique called blue native PAGE (BN-PAGE), first described by Sch&#228;gger et al.25, is superior in terms of resolution. It was successfully used to determine the molecular weights of large native protein complexes from various organelles or from cellular lysates and their relative abundances26,27.
To further elucidate the complex composition of protein complexes, it is necessary to separate the individual components under denaturing conditions, leading to complete disassembly of the complex components. This can be achieved by a second dimension of SDS-PAGE 28,29 or, to achieve higher resolution, by a second dimension of isoelectric focusing (IEF) followed by a third dimension of SDS-PAGE30 and subsequent identification of the proteins using mass spectrometry.
In this protocol, we describe sampling of pure phloem sap from B. napus plants and the analysis of protein complexes from such phloem samples using a 3D electrophoretic approach combining BN-PAGE with Tris-Tricine-urea-PAGE and SDS-PAGE. The separated protein complex components are subsequently identified using mass spectrometry. In addition, we introduce blue native northern blotting as a method allowing the detection of RNAs in large RNP complexes4, extending the applicability of the approach.

<table><tbody><tr><td>1.5 mL reaction tubes</td><td>Eppendorf</td><td>30120086</td><td></td></tr><tr><td>10 well comb for SDS PAGE, thickness 1 mm</td><td>BioRad</td><td>1653359</td><td></td></tr><tr><td>2-Propanol</td><td>AppliChem</td><td>131090</td><td></td></tr><tr><td>30 % Acrylamide-bisacrylamide solution (29:1) solution</td><td>BioRad</td><td>1610156</td><td></td></tr><tr><td>30 % Acrylamide-bisacrylamide solution (37.5:1) solution</td><td>BioRad</td><td>1610158</td><td></td></tr><tr><td>96 % Ethanol</td><td>Carl Roth</td><td>P075</td><td></td></tr><tr><td>Acetic acid</td><td>Carl Roth</td><td>6755</td><td></td></tr><tr><td>Agarose</td><td>Lonza</td><td>50004</td><td></td></tr><tr><td>Aluminum sulfate-(14-18)-hydrate</td><td>AppliChem</td><td>141101</td><td></td></tr><tr><td>Ammonium peroxodisulfate (APS)</td><td>Carl Roth</td><td>9592.3</td><td></td></tr><tr><td>Bis-Tris</td><td>AppliChem</td><td>A3992</td><td></td></tr><tr><td>Boric acid</td><td>AppliChem</td><td>A2940</td><td></td></tr><tr><td>Bromophenol blue</td><td>AppliChem</td><td>A2331</td><td></td></tr><tr><td>Centrifugal concentrators &lt;em&gt;e.g.&lt;/em&gt; Vivaspin 500 (Mwco 10 kDa)</td><td>Sartorius</td><td>VS0102</td><td></td></tr><tr><td>Chemiluminescent Nucleic Acid Detection Module Kit</td><td>Thermo Fisher Scientific</td><td>89880</td><td></td></tr><tr><td>Chromatography papers &lt;em&gt;e.g.&lt;/em&gt; 3 mm CHR</td><td>Whatman</td><td>3030-153</td><td></td></tr><tr><td>CoolBox M30 System</td><td>Biocision</td><td>BCS-133</td><td></td></tr><tr><td>Coomassie brilliant Blue G-250</td><td>AppliChem</td><td>A3480</td><td></td></tr><tr><td>Diethyl pyrocarbonate (DEPC)</td><td>AppliChem</td><td>A0881</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>AppliChem</td><td>A1101</td><td></td></tr><tr><td>DNase I</td><td>AppliChem</td><td>A3778</td><td></td></tr><tr><td>Ethanol abs.</td><td>AppliChem</td><td>A3693</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>AppliChem</td><td>A1103</td><td></td></tr><tr><td>Gel Imaging system &lt;em&gt;e.g.&lt;/em&gt; Chemidoc Touch</td><td>BioRad</td><td>1708370</td><td></td></tr><tr><td>GeneRuler 1kb plus</td><td>Thermo Fisher Scientific</td><td>SM1331</td><td></td></tr><tr><td>Glycerol</td><td>AppliChem</td><td>141339</td><td></td></tr><tr><td>Glycine</td><td>AppliChem</td><td>131340</td><td></td></tr><tr><td>Heating block TS1</td><td>Biometra</td><td>846-051-500</td><td></td></tr><tr><td>Hybridization oven &lt;em&gt;e.g.&lt;/em&gt; GFL Hybridization Incubator 7601</td><td>GFL</td><td>7601</td><td></td></tr><tr><td>Hypodermic needle (0.8 mm)</td><td>B Braun</td><td>4658309</td><td></td></tr><tr><td>IPG gel comb, thickness 1 mm</td><td>BioRad</td><td>1653367</td><td></td></tr><tr><td>Labeling of RNA &lt;em&gt;e.g.&lt;/em&gt;Biotin 3&#039;end DNA Labeling Kit</td><td>Thermo Fisher Scientific</td><td>89818</td><td></td></tr><tr><td>Native gradient gel &lt;em&gt;e.g.&lt;/em&gt; Novex NativePAGE 4&amp;ndash;16% Bis-Tris protein gel</td><td>Invitrogen</td><td>BN1002BOX</td><td></td></tr><tr><td>Nylon membrane &lt;em&gt;e.g.&lt;/em&gt; Hybond-N+</td><td>Amersham Pharmacia</td><td>RPN203B</td><td></td></tr><tr><td>Ortho-phosphoric acid</td><td>Carl Roth</td><td>6366.1</td><td></td></tr><tr><td>PageRuler prestained protein ladder</td><td>Thermo Fisher Scientific</td><td>26616</td><td></td></tr><tr><td>Power supply for Gel electrophoresis EPS</td><td>Amersham Pharmacia</td><td>18-1130-01</td><td>available from GE Healthcare</td></tr><tr><td>RNA Cleanup Kit</td><td>Qiagen</td><td>74204</td><td></td></tr><tr><td>Semi dry blot &lt;em&gt;e.g&lt;/em&gt;. Fastblot 43B semi dry Blot</td><td>Biometra</td><td>846-015-100</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>AppliChem</td><td>A1149</td><td></td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>AppliChem</td><td>142363</td><td></td></tr><tr><td>Synthesis of cDNA &lt;em&gt;e.g.&lt;/em&gt; RevertAid First Strand cDNA Synthesis Kit</td><td>Thermo Fisher Scientific</td><td>K1621</td><td></td></tr><tr><td>Tetramethylethylenediamine (TEMED)</td><td>AppliChem</td><td>A1148</td><td></td></tr><tr><td>Tricine</td><td>AppliChem</td><td>A3954</td><td></td></tr><tr><td>Tris</td><td>AppliChem</td><td>A1086</td><td></td></tr><tr><td>Tri-sodium citrate dihydrate</td><td>AppliChem</td><td>A3901</td><td></td></tr><tr><td>TRIzol Reagent</td><td>Thermo Fisher Scientific</td><td>15596026</td><td></td></tr><tr><td>ULTRAhyb Ultrasensitive Hybridization Buffer</td><td>Thermo Fisher Scientific</td><td>AM8670</td><td></td></tr><tr><td>Urea</td><td>AppliChem</td><td>A1360</td><td></td></tr><tr><td>UV Crosslinker &lt;em&gt;e.g.&lt;/em&gt; Stratalinker 2400</td><td>Agilent</td><td>NC0477671</td><td>from Fisher Scientific</td></tr><tr><td>Vertical electrophoresis apparatus &lt;em&gt;e.g.&lt;/em&gt;The XCell SureLock Mini-Cell or Mini-Protean III System</td><td>Thermo Fisher Scientific or BioRad</td><td>EI0001 or 1658004</td><td></td></tr><tr><td>Wipers &lt;em&gt;e.g.&lt;/em&gt; KIMWIPES Delicate Task Wipers</td><td>Kimberly-Clark</td><td>34120</td><td></td></tr></tbody></table>

phloem sap sampling from brassica napus for 3d-page of protein and ribonucleoprotein complexes

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

Watch this Scientific Journal Video about Phloem Sap Sampling from Brassica napus for 3D-PAGE of Protein and Ribonucleoprotein Complexes at JoVE.com

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

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

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

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

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

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13.7K Views.  Universität Hamburg. Here we present a protocol to analyze the protein composition of large native protein:protein and protein:nucleic acid complexes from oilseed rape (B. napus) phloem exudate using a 3D polyacrylamide gel electrophoresis (PAGE) approach combining blue native (BN) with two denaturing PAGEs followed by mass spectrometric identification.

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

mRNA Interactome Capture from Plant Protoplasts

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

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

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

Biochemistry

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+

In recent years, the carbohydrate moieties of plants have received considerable attention, as they are a potential source of cross-reactive, allergy-provoking immune responses. In addition, carbohydrate structures also play a critical role in plant metabolism. Here, we present a simple and rapid method for preparing and analyzing N-glycans from different cultivars of radish (Raphanus sativus) using an N-glycanase specific for the release of plant-derived carbohydrate structures. To achieve this, crude trichloroacetic acid precipitates of radish homogenates were treated with PNGase H+, and labeled using 2-aminobenzamide as a fluorescent tag. The labeled N-glycan samples were subsequently analyzed by ultra performance liquid chromatography (UPLC) separation and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for a detailed structural evaluation and to quantify relative abundancies of the radish-derived N-glycan structures. This protocol can also be used for the analysis of N-glycans from various other plant species, and may be useful for further investigation of the function and effects of N-glycans on human health.

Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+

We describe a simple and rapid method for the preparation and analysis of N-glycans from different cultivars of radish (Raphanus sativus).

In recent years, the carbohydrate moieties of plants have received considerable attention, as they are a potential source of cross-reactive, ...

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed complexes. The latter can be elegantly resolved according to molecular size using native polyacrylamide gel electrophoresis (BN-PAGE). Coupling of such separations to sensitive mass spectrometry (BN-MS) has been well-established and theoretically allows for exhaustive assessment of the extractable complexome in biological samples. However, this approach is rather laborious and provides limited complex size resolution and sensitivity. Also, its application has remained restricted to abundant mitochondrial and plastid proteins. Thus, for a majority of proteins, information regarding integration into stable protein complexes is still lacking. Presented here is an optimized approach for complexome profiling comprising preparative-scale BN-PAGE separation, sub-millimeter sampling of broad gel lanes by cryomicrotome slicing, and mass spectrometric analysis with label-free protein quantification. The procedures and tools for critical steps are described in detail. As an application, the report describes complexome analysis of a solubilized endosome-enriched membrane fraction from mouse kidneys, with 2,545 proteins profiled in total. The results demonstrate identification of uniform, low-abundance membrane proteins such as intracellular ion channels as well as high resolution, complex protein assembly patterns, including glycosylation isoforms. The results are in agreement with independent biochemical analyses. In summary, this methodology allows for comprehensive and unbiased identification of protein (super)complexes and their subunit composition, providing a basis for investigating stoichiometry, assembly, and interaction dynamics of protein complexes in any biological system.

A versatile cryoslicing BN-MS protocol using a microtome is presented for high-resolution complexome profiling.

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed ...

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

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

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

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

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

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

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

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

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

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

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

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

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

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

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

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

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

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Chapters in this video

mRNA Interactome Capture from Plant Protoplasts

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H⁺

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

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

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