This work was funded by National Natural Science Foundation of China (31671257) and Hubei Collaborative Innovation Center for Grain Industry (LXT-16-18).

1. Arabidopsis vertical growth in MS medium
<ol>
	<li>The interior of laminar flow cabinet needs to be treated with 30 min UV light and 15 min airflow before usage. Make sure to close the glass door when UV light is on. Spray all tools and plates with 70% ethanol before placing them in the cabinet.</li>
	<li>Prepare the Murashige and Skoog (MS) medium in a standard 90 mm-diameter Petri dish under a laminar flow cabinet. 
	NOTE: The MS medium contains the following components: 20.6 mM NH4NO...

<ol>
	<li>Grignon, N., Touraine, B., Durand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=6(5)Carboxyfluorescein+as+a+tracer+of+phloem+sap+translocation.">6(5)Carboxyfluorescein as a tracer of phloem sap translocation.</a> American Journal of Botany. 76, 871-877 (1989).</li><li>Wright, K. M., 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=The+fluorescent+probe+HPTS+as+a+phloem-mobile,+symplastic+tracer:+an+evaluation+using+confocal+laser+scanning+microscopy.">The fluorescent probe HPTS as a phloem-mobile, symplastic tracer: an evaluation using confocal laser scanning microscopy.</a> Journal of Experimental Botany. 47 (3), 439-445 (1996).</li><li>Oparka, K. J., Prior, 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=Movement+of+Lucifer+Yellow+CH+in+potato+tuber+storage+tissues:+A+comparison+of+symplastic+and+apoplastic+transport.">Movement of Lucifer Yellow CH in potato tuber storage tissues: A comparison of symplastic and apoplastic transport.</a> Planta. 176 (4), 533-540 (1988).</li><li>Knoblauch, 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=Multispectral+Phloem-Mobile+Probes:+Properties+and+Applications.">Multispectral Phloem-Mobile Probes: Properties and Applications.</a> Plant Physiology. 167 (4), 1211-1220 (2015).</li><li>Oparka, K. J., Duckett, C. M., Prior, D. A. M., Fisher, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+of+phloem+unloading+in+the+root+tip+of+Arabidopsis.">Real-time imaging of phloem unloading in the root tip of Arabidopsis.</a> The Plant Journal. 6 (5), 759-766 (1994).</li><li>Viola, 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=Tuberization+in+Potato+Involves+a+Switch+from+Apoplastic+to+Symplastic+Phloem+Unloading.">Tuberization in Potato Involves a Switch from Apoplastic to Symplastic Phloem Unloading.</a> The Plant Cell. 13 (2), 385-398 (2001).</li><li>Roberts, A. 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=Phloem+Unloading+in+Sink+Leaves+of+Nicotiana+benthamiana:+Comparison+of+a+Fluorescent+Solute+with+a+Fluorescent+Virus.">Phloem Unloading in Sink Leaves of Nicotiana benthamiana: Comparison of a Fluorescent Solute with a Fluorescent Virus.</a> The Plant Cell. 9 (8), 1381-1396 (1997).</li><li>P&#233;ron, T., 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=New+Insights+into+Phloem+Unloading+and+Expression+of+Sucrose+Transporters+in+Vegetative+Sinks+of+the+Parasitic+Plant+Phelipanche+ramosa+L+(Pomel).">New Insights into Phloem Unloading and Expression of Sucrose Transporters in Vegetative Sinks of the Parasitic Plant Phelipanche ramosa L (Pomel).</a> Frontiers in Plant Science. 7 (2048), (2017).</li><li>Spallek, T., 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=Interspecies+hormonal+control+of+host+root+morphology+by+parasitic+plants.">Interspecies hormonal control of host root morphology by parasitic plants.</a> Proceedings of the National Academy of Sciences of the USA. 114 (20), 5283-5288 (2017).</li><li>Complainville, 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=Nodule+initiation+involves+the+creation+of+a+new+symplasmic+field+in+specific+root+cells+of+medicago+species.">Nodule initiation involves the creation of a new symplasmic field in specific root cells of medicago species.</a> The Plant Cell. 15 (12), 2778-2791 (2003).</li><li>Bederska, M., Borucki, W., Znojek, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Movement+of+fluorescent+dyes+Lucifer+Yellow+(LYCH)+and+carboxyfluorescein+(CF)+in+Medicago+truncatula+Gaertn.+roots+and+root+nodules.">Movement of fluorescent dyes Lucifer Yellow (LYCH) and carboxyfluorescein (CF) in Medicago truncatula Gaertn. roots and root nodules.</a> Symbiosis. 58 (1-3), 183-190 (2012).</li><li>Robards, A. W., 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=Plasmodesmata.">Plasmodesmata.</a> Annual Review of Plant Physiology and Plant Molecular Biology. 41 (1), 369-419 (1990).</li><li>Roberts, A. G., 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=Plasmodesmata+and+the+control+of+symplastic+transport.">Plasmodesmata and the control of symplastic transport.</a> Plant, Cell &amp; Environment. 26 (1), 103-124 (2003).</li><li>Duckett, C. M., Oparka, K. J., Prior, D. A. M., Dolan, L., Roberts, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dye-coupling+in+the+root+epidermis+of+Arabidopsis+is+progressively+reduced+during+development.">Dye-coupling in the root epidermis of Arabidopsis is progressively reduced during development.</a> Development. 120 (11), 3247-3255 (1994).</li><li>Palevitz, B. A., Hepler, P. K. <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+dye+coupling+of+stomatal+cells+of+Allium+and+Commelina+demonstrated+by+microinjection+of+Lucifer+yellow.">Changes in dye coupling of stomatal cells of Allium and Commelina demonstrated by microinjection of Lucifer yellow.</a> Planta. 164 (4), 473-479 (1985).</li><li>van Bel, A. J. E., Kempers, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symplastic+isolation+of+the+sieve+element-companion+cell+complex+in+the+phloem+of+Ricinus+communis+and+Salix+alba+stems.">Symplastic isolation of the sieve element-companion cell complex in the phloem of Ricinus communis and Salix alba stems.</a> Planta. 183 (1), 69-76 (1991).</li><li>Erwee, M. G., Goodwin, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symplast+domains+in+extrastelar+tissues+of+Egeria+densa+Planch.">Symplast domains in extrastelar tissues of Egeria densa Planch.</a> Planta. 163 (1), 9-19 (1985).</li><li>Oparka, K. J., Prior, D. A. M., Wright, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symplastic+communication+between+primary+and+developing+lateral+roots+of+Arabidopsis+thaliana.">Symplastic communication between primary and developing lateral roots of Arabidopsis thaliana.</a> Journal of Experimental Botany. 46 (2), 187-197 (1995).</li><li>Christensen, N. M., Faulkner, C., Oparka, K. <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+Unidirectional+Flow+through+Plasmodesmata.">Evidence for Unidirectional Flow through Plasmodesmata.</a> Plant Physiology. 150 (1), 96-104 (2009).</li><li>Wr&#243;bel-Marek, J., Kurczy&#324;ska, E., P&#322;achno, B. J., Kozieradzka-Kiszkurno, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+symplasmic+domains+in+the+embryo+and+seed+of+Sedum+acre+L.+(Crassulaceae).">Identification of symplasmic domains in the embryo and seed of Sedum acre L. (Crassulaceae).</a> Planta. 245 (3), 491-505 (2017).</li><li>Liang, D., White, R. G., Waterhouse, 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=Gene+silencing+in+Arabidopsis+spreads+from+the+root+to+the+shoot,+through+a+gating+barrier,+by+template-dependent,+non-vascular,+cell+to+cell+movement.">Gene silencing in Arabidopsis spreads from the root to the shoot, through a gating barrier, by template-dependent, non-vascular, cell to cell movement.</a> Plant Physiology. 159 (3), 984-1000 (2012).</li><li>Radford, J. E., White, R. G. <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+tissue-preparation-induced+callose+synthesis+on+estimates+of+plasmodesma+size+exclusion+limits.">Effects of tissue-preparation-induced callose synthesis on estimates of plasmodesma size exclusion limits.</a> Protoplasma. 216 (1-2), 47-55 (2001).</li><li>Kollist, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Responses+to+Abiotic+Stress:+Priming+the+Landscape+for+the+Signal+Transduction+Network.">Rapid Responses to Abiotic Stress: Priming the Landscape for the Signal Transduction Network.</a> Trends in Plant Science. 24 (1), 25-37 (2019).</li><li>Haupt, S., Duncan, G. H., Holzberg, S., Oparka, K. 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Emerging studies have shown that plants can rapidly respond to external stimuli23, including manipulation introduced to the experimental procedures22. In our initial experiment, our oversight of this knowledge often leads to staining failure. With these lessons, we suggest that the following precautions should be kept in mind when performing this experiment: (1) the seeds after harvest should be kept in a storage cabinet set to a low temperature and low moisture; (2) manipu...

Many fluorescent tracers with a range of spectral properties, such as 5(6)-carboxyfluorescein (CF)1, 8-hydroxypyrene-1,3,6-trisulphonic acid2, Lucifer yellow CH (LYCH)3, Esculin and CTER4, have been developed and applied in plants to monitor symplastic movement and phloem activity. Generally, a symplastic dye is loaded into a cut in the target tissue and the sequential dispersion of the reporter into other parts of plant will demonstrate the intercellular communication. Although the mechanism of dye absorption is not fully understood, the principle underlying CF movement inside live cells has been widely acknowledged. The ester form of CF (CF diacetate, CFDA) is non-fluorescent, but membrane-permeable. This property allows rapid membrane diffusion of the dye into cells. Once inside live cells, intracellular esterases remove the acetate groups at the 3&#39; and 6&#39; position of CFDA, releasing the fluorescent and membrane-impermeable CF (Figure 1, alternatively refer Wright et al.2); CF can then move through the plasmodesmata to other parts of plants.
A well-established procedure with CFDA is that it can be loaded into source leaves and used to monitor the phloem streaming and phloem unloading in the sink tissues of many species, e.g., as CF unloading in the Arabidopsis root5, phloem unloading during potato tuberization6, phloem unloading in the Nicotiana sink leaves7, and so on. By similar loading approaches, other studies have adopted this dye to demonstrate the symplastic connection between host and parasite8,9, or to reveal the symbiotic relationships10,11.
Another way to make use of this dye is to load it into specific cells or single cell by microinjection to determine its distribution pattern. Such sophisticated techniques have greatly facilitated our deeper understanding of plasmodesmata-mediated intercellular communication, particularly in the development of the concept of symplastic domain12,13. For example, the microinjection of CFDA into cotyledon cells of Arabidopsis resulted in the dye-coupling pattern in the hypocotyl epidermis but non-coupling in the underlying cells or in the root epidermis, therefore the hypocotyl epidermis forms a symplastic domain14. Similar domains, such as the stomatal guard cells15, sieve element-companion cells16, root hair cells14 and root cap17,18 have been identified by microinjection technique. Most surprisingly, some domains allow tracer molecules to move in a certain direction. Take the trichome domain for example, microinjection of a fluorescent probe into the supporting epidermal cell leads to the flow of tracer into the trichome domain, however, the reverse injection does not hold true19. A recent report has also found similar situations in the symplastic domains of the Sedum embryo20. Thus, all above cases imply that swapping of loading sites may lead to novel insights into symplastic communication. Our previous experiment aiming to dissect the route of root-to-shoot mobile silencing identified a novel symplastic domain, or the HEJ (Hypocotyl-epicotyl junction) zone, which was further verified through the root-loading (non-canonical sink-loading) CFDA experiment21. Here, we further elaborate the root-to-shoot CF movement by using a simple method and recover a potential symplastic domain by shifting the loading sites. Furthermore, this procedure may be adapted to differentiate genetic backgrounds that have altered root-to-shoot long-distance transport.

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			<td height="24" style="height:24px;width:223px;">KNO3&#160;&#160;</td>
			<td style="width:153px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">10017218</td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">KH2PO4&#160;</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10017608</td>
			<td></td>
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		<tr height="24">
			<td height="24" style="height:24px;">MgSO4&#183;7H2O</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10013018</td>
			<td></td>
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		<tr height="24">
			<td height="24" style="height:24px;">CaCl2&#183;2H2O</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>20011160</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">MnSO4&#183;H2O&#160;</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10013418</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Na2MoO4&#183;2H2O</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10019818</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Boric Acid</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10004818</td>
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		<tr height="24">
			<td height="24" style="height:24px;">ZnSO4&#183;7H2O</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10024018</td>
			<td></td>
		</tr>
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			<td height="24" style="height:24px;">CuSO4&#183;5H2O</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10008218</td>
			<td></td>
		</tr>
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			<td height="24" style="height:24px;">CoCl2&#183;6H2O</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10007216</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">KI</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10017160</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">FeSO4&#183;7H2O</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10012118</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA&#160;</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10009717</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaOH</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10019718</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KOH</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10017018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10021418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Myo-inositol&#160;&#160;</td>
			<td>MACKLIN</td>
			<td>I811835</td>
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			<td height="21" style="height:21px;">Nicotinic Acid&#160;</td>
			<td>MACKLIN</td>
			<td>N814565</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Pyridoxine HCl</td>
			<td>MACKLIN</td>
			<td>V820447</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thiamine HCl</td>
			<td>MACKLIN</td>
			<td>T818865</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycine</td>
			<td>MACKLIN</td>
			<td>G800880</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Agar powder</td>
			<td>Novon</td>
			<td>ZZ14022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescence Microscope</td>
			<td>Zeiss</td>
			<td>Axio Zoom V16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting microscope</td>
			<td>SDPTOP</td>
			<td>SRE-1030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200&#956;l pipette</td>
			<td>Dragon Laboratory Instruments</td>
			<td colspan="2">713111110000-20-200ul</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2.5&#956;l pipette</td>
			<td>Eppendorf</td>
			<td>3120000011</td>
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			<td height="21" style="height:21px;">Fine forceps&#160;</td>
			<td>TWEEZERS</td>
			<td>ST-15</td>
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			<td height="21" style="height:21px;">Parafilm</td>
			<td>PARAFILM</td>
			<td>PM-996</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Stainless steel double-sided blade</td>
			<td>Gillette</td>
			<td colspan="2">Platinum-Plus Double-Edge Blade</td>
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shootward movement of cfda tracer loaded in the bottom sink tissues of arabidopsis

The symplastic tracer 5(6)-carboxyfluorescein diacetate (CFDA) has been widely applied in living plants to demonstrate the intercellular connection, phloem transport and vascular patterning. This protocol shows bottom-to-top carboxyfluorescein (CF) movement in the Arabidopsis by using the root-cutting and the hypocotyl-pinching procedure respectively. These two different procedures result in different efficiencies of CF movement: about 91% appearance of CF in the shoots with the hypocotyl-pinching procedure, whereas only about 70% appearance of CF with the root-cutting procedure. The simple change of loading sites, resulting in significant changes in the mobile efficiency of this symplastic dye, suggests CF movement might be subject to the symplastic regulation, most probably by the root-hypocotyl junction.

Symplastic movement is often subject to environmental fluctuations. Perturbation of the plant growing state, and even the process of tissue preparation will affect the size exclusion limit of plasmodesmata, thus affecting the symplastic transport22. To improve the staining efficiency, we confine our operation in the growth room, where the temperature and moisture is tightly controlled, and also perform the whole procedure as quickly as possible (ideally within 10-1...

Watch this Scientific Journal Video about Shootward Movement of CFDA Tracer Loaded in the Bottom Sink Tissues of Arabidopsis at JoVE.com

Shootward Movement of CFDA Tracer Loaded in the Bottom Sink Tissues of Arabidopsis

The goal of this protocol is to show how to load the CFDA into different sites of the bottom parts of Arabidopsis. We then present the resulting distribution pattern of CF in the shoots.

Shootward Movement of CFDA Tracer Loaded in the Bottom Sink Tissues of Arabidopsis

The symplastic tracer 5(6)-carboxyfluorescein diacetate (CFDA) has been widely applied in living plants to demonstrate the intercellular connection, ...

The widely-used probe CFDA, or short for carboxyfluorescein diacetate, is non-fluorescent. The two H3 groups and the 3'6'position on this compound render it membrane-permeant. Where once inside cells, internal cellular acetates will remove the acetate groups and release the new form of CFDA.That's a carboxyfluorescein, or the CF, which is fluorescent. And because this new form is less membrane-permeant, the weight, all the cells is to move through the intercellular pores, or the plasmodesmata in plants, that's making this diacetate the ideal probe to study intercellular transport in phloem activity. In today's video, we're going to demonstrate the CFD loading into the root and hypocotyl of the Arabidopsis plants, respectively, and we will show how the two slightly different procedures will affect the bottom to top movement of this fluorescent probe.Achieve staining results consistently, we store the harvested Arabidopsis seeds in four Celsius degree, 40%moisture cabinet. Newly harvested seeds stored in this condition for more than seven days are readily used for sowing. To ensure every plant is grown in a relatively similar space in the Petri dish, we are using a seed zone grade card.Now, wetting the sterilized tip, dip in the sterilized seeds, and sow the seeds one-by-one on the designated position. After finish sowing, seal the Petri dish with parafilm and a sticky tape, put them on a Petri dish stand to let it grow vertically. Here are the tools we are going to use in this experiment.Please note that we'd better load the dye as quickly as we can. Normally, you finish the whole process in 10 to 15 minutes. Before we perform the CFDA loading experiment, we always prepare fresh CFDA solution.Here, dilute the one millimolar CFDA into five micromolar concentration with water before immediately use. Cut the parafilm into small pieces in three by three millimeter size. Because there is condensation on the lid and bottom of the Petri dish, we need to clear them out with paper towel to avoid dye flowing around.A small parafilm piece is put under the root. Lift the plants and put them on the lid. Cut the roots about five to 10 millimeter below the hypocotyl.Take the shoots back on the small pieces of parafilm. Now carefully apply one microliter CFDA onto the cutting end of the root. Try to avoid contaminating all the proto-plants.Then cover the lid, and then leave them in the growth room for two hours. The Hypocotyl-pinching Method. The small parafilm piece, as previously prepared, is put under the root-hypocotyl junction.We use a forecep to gently pinch the hypocotyl close to the root tissue, and carefully apply 0.1 microliter CFDA onto the pinched side and leave the plant for two hours. The photos from nine to 12 day after zone staining plants. They all show the vascular staining pattern in both the cotyledons and the true leaves.Only in a very rare case, the cotyledon leaf showed a half-leaf staining pattern. So basically, the two methods did not make a difference in the CF staining pattern, however if we perform dye loading into more and more plants, we find the two procedures give out a significant difference deal in efficiency. With the root-cut method, about 70%of the plants show the fluorescent signal in the shoot.But the efficiency can be increased to 91%using the hypocotyl-pinching method. We also tried the root-pinching method, but this method result in the even lower efficiency, it's about 30%Therefore, it seems the way to load the dye does not account for the staining difference. The difference most probably lies in the loading sites, which might be separated by a symplastic barrier.We showed how we load the CFDA into the Arabidopsis roots and hypocotyl. We used two slightly different procedures, and these two slightly different procedures result in significant difference in this bottom to top probe movement. We suggest that this method could help us to identify a potential symplastic barrier for root-derived shootward signals.

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6.5K vistas.  Yangtze University. La sonda ampliamente utilizada CFDA, o abreviatura de diacetato de carboxicfluoresceína, no es fluorescente. Los dos grupos H3 y la posición de 3'6' en este compuesto lo hacen permeante de membrana. Donde una vez dentro de las células, los acetatos celulares internos eliminarán los grupos de acetato y liberarán la nueva forma de CFDA.Eso es una carboxifluoresceína, o el CF, que es fluorescente. Y debido a que esta nueva forma es menos permeante de membrana, el peso, todas las cé...