Name: shootward movement of cfda tracer loaded in the bottom sink tissues of arabidopsis
Uploaded: 2019-05-11T15:00:00
Description: Watch this Scientific Journal Video about Shootward Movement of CFDA Tracer Loaded in the Bottom Sink Tissues of Arabidopsis at JoVE.com

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>Sinopharm Chemical Reagent</td>
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			<td height="24" style="height:24px;">MgSO4&#183;7H2O</td>
			<td>Sinopharm Chemical Reagent</td>
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			<td height="24" style="height:24px;">CaCl2&#183;2H2O</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>20011160</td>
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			<td height="24" style="height:24px;">MnSO4&#183;H2O&#160;</td>
			<td>Sinopharm Chemical Reagent</td>
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			<td>Sinopharm Chemical Reagent</td>
			<td>10019818</td>
			<td></td>
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			<td height="21" style="height:21px;">Boric Acid</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10004818</td>
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			<td height="24" style="height:24px;">ZnSO4&#183;7H2O</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10024018</td>
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			<td height="24" style="height:24px;">CuSO4&#183;5H2O</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10008218</td>
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			<td height="24" style="height:24px;">CoCl2&#183;6H2O</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10007216</td>
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			<td height="21" style="height:21px;">KI</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10017160</td>
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			<td>Sinopharm Chemical Reagent</td>
			<td>10012118</td>
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			<td height="21" style="height:21px;">EDTA&#160;</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10009717</td>
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			<td height="21" style="height:21px;">NaOH</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10019718</td>
			<td></td>
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			<td height="21" style="height:21px;">KOH</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10017018</td>
			<td></td>
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			<td height="21" style="height:21px;">Sucrose</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10021418</td>
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			<td height="21" style="height:21px;">Myo-inositol&#160;&#160;</td>
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			<td>I811835</td>
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			<td>MACKLIN</td>
			<td>N814565</td>
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			<td height="21" style="height:21px;">Pyridoxine HCl</td>
			<td>MACKLIN</td>
			<td>V820447</td>
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			<td height="21" style="height:21px;">Thiamine HCl</td>
			<td>MACKLIN</td>
			<td>T818865</td>
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			<td height="21" style="height:21px;">Glycine</td>
			<td>MACKLIN</td>
			<td>G800880</td>
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			<td height="21" style="height:21px;">Agar powder</td>
			<td>Novon</td>
			<td>ZZ14022</td>
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			<td height="21" style="height:21px;">Fluorescence Microscope</td>
			<td>Zeiss</td>
			<td>Axio Zoom V16</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Dissecting microscope</td>
			<td>SDPTOP</td>
			<td>SRE-1030</td>
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			<td height="21" style="height:21px;">200&#956;l pipette</td>
			<td>Dragon Laboratory Instruments</td>
			<td colspan="2">713111110000-20-200ul</td>
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			<td height="21" style="height:21px;">2.5&#956;l pipette</td>
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			<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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			<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.

shootward-movement-cfda-tracer-loaded-bottom-sink-tissues

Research

JoVE Journal

Environment

Technique for Studying Arthropod and Microbial Communities within Tree Tissues

Phloem tissues of pine are habitats for many thousands of organisms. Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear young, feed, or hide from natural enemies or harsh environmental conditions outside of the tree. Organisms that persist within the phloem habitat are difficult to observe given their location under bark. We provide a technique to preserve intact phloem and prepare it for experimentation with invertebrates and microorganisms. The apparatus is called a &#8216;phloem sandwich&#8217; and allows for the introduction and observation of arthropods, microbes, and other organisms. This technique has resulted in a better understanding of the feeding behaviors, life-history traits, reproduction, development, and interactions of organisms within tree phloem. The strengths of this technique include the use of inexpensive materials, variability in sandwich size, flexibility to re-open the sandwich or introduce multiple organisms through drilled holes, and the preservation and maintenance of phloem integrity. The phloem sandwich is an excellent educational tool for scientific discovery in both K-12 science courses and university research laboratories.

We provide a technique to preserve intact tree phloem and prepare it for observation. We create an apparatus called a phloem sandwich that allows for the introduction and observation of arthropods, microbes, and other organisms that inhabit phloem tissues.

Phloem tissues of pine are habitats for many thousands of organisms.  Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear ...

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

Use of Autometallography to Localize and Semi-Quantify Silver in Cetacean Tissues

Silver nanoparticles (AgNPs) have been extensively used in commercial products, including textiles, cosmetics, and health care items, due to their strong antimicrobial effects. They also may be released into the environment and accumulate in the ocean. Therefore, AgNPs are the major source of Ag contamination, and public awareness of the environmental toxicity of Ag is increasing. Previous studies have demonstrated the bioaccumulation (in producers) and magnification (in consumers/predators) of Ag. Cetaceans, as the apex predators of ocean, may have been negatively affected by the Ag/Ag compounds. Although the concentrations of Ag/Ag compounds in cetacean tissues can be measured by inductively coupled plasma mass spectroscopy (ICP-MS), the use of ICP-MS is limited by its high capital cost and the requirement for tissue storage/preparation. Therefore, an autometallography (AMG) method with an image quantitative analysis by using formalin-fixed, paraffin-embedded (FFPE) tissue may be an adjuvant method to localize Ag distribution at the suborgan level and estimate the Ag concentration in cetacean tissues. The AMG positive signals are mainly brown to black granules of various sizes in the cytoplasm of proximal renal tubular epithelium, hepatocytes, and Kupffer cells. Occasionally, some amorphous golden yellow to brown AMG positive signals are noted in the lumen and basement membrane of some proximal renal tubules. The assay for estimating the Ag concentration is named the Cetacean Histological Ag Assay (CHAA), which is a regression model established by the data from image quantitative analysis of the AMG method and ICP-MS. The use of AMG with CHAA to localize and semi-quantify heavy metals provides a convenient methodology for spatio-temporal and cross-species studies.

A protocol is presented to localize Ag in cetacean liver and kidney tissues by autometallography. Furthermore, a new assay, named the cetacean histological Ag assay (CHAA) is developed to estimate the Ag concentrations in those tissues.

Silver nanoparticles (AgNPs) have been extensively used in commercial products, including textiles, cosmetics, and health care items, due to their strong ...

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

Stomatal movement mediates plant gas exchange, which is essential for photosynthesis and transpiration. Stomatal opening and closing are accomplished by a significant increase and decrease in guard cell volume, respectively. Because shuttle transport of ions and water occurs between guard cells and larger neighboring epidermal cells during stomatal movement, the spaced distribution of plant stomata is considered an optimal distribution for stomatal movement. Experimental systems for perturbing the spaced pattern of stomata are useful to examine the spacing pattern's significance. Several key genes associated with the spaced stomatal distribution have been identified, and clustered stomata can be experimentally induced by altering these genes. Alternatively, clustered stomata can be also induced by exogenous treatments without genetic modification. In this article, we describe a simple induction system for clustered stomata in Arabidopsis thaliana seedlings by immersion treatment with a sucrose-containing medium solution. Our method is easy and directly applicable to transgenic or mutant lines. Larger chloroplasts are presented as a cell biological hallmark of sucrose-induced clustered guard cells. In addition, a representative confocal microscopic image of cortical microtubules is shown as an example of intracellular observation of clustered guard cells. The radial orientation of cortical microtubules is maintained in clustered guard cells as in spaced guard cells in control conditions.

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

The goal of this protocol is to demonstrate how to induce clustered stomata in cotyledons of Arabidopsis thaliana seedlings by immersion treatment with a sugar-containing medium solution and how to observe intracellular structures such as chloroplasts and microtubules in the clustered guard cells using confocal laser microscopy.

Stomatal movement mediates plant gas exchange, which is essential for photosynthesis and transpiration. Stomatal opening and closing are accomplished by a ...

Fluorescently Labeled Bacteria as a Tracer to Reveal Novel Pathways of Organic Carbon Flow in Aquatic Ecosystems

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial communities has many limitations, and determining a predator, prey, and predatory rates is often difficult. Presented here is an optimized method based on the addition of fluorescently labelled prey as a tracer, which allows for reliable quantitation of the grazing rates in aquatic predatory eukaryotes and estimation of nutrient transfer to higher trophic levels.

Presented here is a protocol for a single-cell, epifluorescence microscopy-based technique to quantify grazing rates in aquatic predatory eukaryotes with high precision and taxonomic resolution.

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial ...

A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant&ndash;Environment Interactions

Food security for the growing global population is a major concern. The data provided by genomic tools far exceeds the supply of phenotypic data, creating a knowledge gap. To meet the challenge of improving crops to feed the growing global population, this gap must be bridged.
Physiological traits are considered key functional traits in the context of responsiveness or sensitivity to environmental conditions. Many recently introduced high-throughput (HTP) phenotyping techniques are based on remote sensing or imaging and are capable of directly measuring morphological traits, but measure physiological parameters mainly indirectly.
This paper describes a method for direct physiological phenotyping that has several advantages for the functional phenotyping of plant&#8211;environment interactions. It helps users overcome the many challenges encountered in the use of load-cell gravimetric systems and pot experiments. The suggested techniques will enable users to distinguish between soil weight, plant weight and soil water content, providing a method for the continuous and simultaneous measurement of dynamic soil, plant and atmosphere conditions, alongside the measurement of key physiological traits. This method allows researchers to closely mimic field stress scenarios while taking into consideration the environment&#8217;s effects on the plants&#8217; physiology. This method also minimizes pot effects, which are one of the major problems in pre-field phenotyping. It includes a feed-back fertigation system that enables a truly randomized experimental design at a field-like plant density. This system detects the soil-water-content limiting threshold (&#952;) and allows for the translation of data into knowledge through the use of a real-time analytic tool and an online statistical resource. This method for the rapid and direct measurement of the physiological responses of multiple plants to a dynamic environment has great potential for use in screening for beneficial traits associated with responses to abiotic stress, in the context of pre-field breeding and crop improvement.

A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant&#8211;Environment Interactions

This high-throughput, telemetric, whole-plant water relations gravimetric phenotyping method enables direct and simultaneous real-time measurements, as well as the analysis of multiple yield-related physiological traits involved in dynamic plant&#8211;environment interactions.

Food security for the growing global population is a major concern. The data provided by genomic tools far exceeds the supply of phenotypic data, creating ...

Using Changes in Leaf Transmission to Investigate Chloroplast Movement in Arabidopsis thaliana

Chloroplast movement in leaves has been shown to help minimize photoinhibition and increase growth under certain conditions. Much can be learned about chloroplast movement by studying the chloroplast positioning in leaves using e.g., confocal fluorescence microscopy, but access to this type of microscopy is limited. This protocol describes a method that uses the changes in leaf transmission as a proxy for chloroplast movement. If chloroplasts are spread out in order to maximize light interception, the transmission will be low. If chloroplasts move towards the anticlinal cell walls to avoid light, the transmission will be higher. This protocol describes how to use a straightforward, home-built instrument to expose leaves to different blue light intensities and quantify the dynamic changes in leaf transmission. This approach allows researchers to quantitatively describe chloroplast movement in different species and mutants, study the effects of chemicals and environmental factors on it, or screen for novel mutants e.g., to identify missing components in the process that leads from light perception to the movement of chloroplasts.

Using Changes in Leaf Transmission to Investigate Chloroplast Movement in Arabidopsis thaliana

Many plant species change the positioning of chloroplasts to optimize light absorption. This protocol describes how to use a straightforward, home-built instrument to investigate chloroplast movement in Arabidopsis thaliana leaves using changes in the transmission of light through a leaf as a proxy.

Chloroplast movement in leaves has been shown to help minimize photoinhibition and increase growth under certain conditions. Much can be learned about ...

Elucidating the Metabolism of 2,4-Dibromophenol in Plants

Crops can be extensively exposed to organic pollutants, since soil is a major sink for pollutants discarded into the environment. This creates potential human exposure through the consumption of pollutant-accumulated foods. Elucidating the uptake and metabolism of xenobiotics in crops is essential for the assessment of dietary exposure risk in humans. However, for such experiments, the use of intact plants requires long-term experiments and complex sample preparation protocols that can be affected by various factors. Plant callus cultures combined with high-resolution mass spectrometry (HRMS) may provide a solution for the accurate and time-saving identification of metabolites of xenobiotics in plants, as it can avoid interference from the microbial or fungal microenvironment, shorten the treatment duration, and simplify the matrix effect of intact plants. 2,4-dibromophenol, a typical flame retardant and endocrine disrupter, was chosen as the model substance due to its widespread occurrence in soil and its uptake potential by plants. Herein, plant callus was generated from asepsis seeds and exposed to sterile 2,4-dibromophenol-containing culture medium. The results showed that eight metabolites of 2,4-dibromophenol were identified in the plant callus tissues after 120 h of incubation. This indicates that 2,4-dibromophenol was rapidly metabolized in&#160;the plant callus tissues. Thus, the plant callus culture platform is an effective method to evaluate the uptake and metabolism of xenobiotics in plants.

The present protocol describes a simple and efficient method for the identification of 2,4-dibromophenol metabolites in plants.

Crops can be extensively exposed to organic pollutants, since soil is a major sink for pollutants discarded into the environment. This creates potential ...

Determination of the Absorption, Translocation, and Distribution of Imidacloprid in Wheat

Neonicotinoids, a class of insecticides, are widely used because of their novel modes of action, high insecticidal activity, and strong root uptake. Imidacloprid, the most widely used insecticide worldwide, is a representative first-generation neonicotinoid and is used in pest control for crops, vegetables, and fruit trees. With such a broad application of imidacloprid, its residue in crops has attracted increasing scrutiny. In the present study, 15 wheat seedlings were placed in a culture medium containing 0.5 mg/L or 5 mg/L imidacloprid for hydroculture. The content of imidacloprid in the wheat roots and leaves was determined after 1 day, 2 days, and 3 days of hydroculture to explore the migration and distribution of imidacloprid in wheat. The results showed that imidacloprid was detected both in the roots and leaves of the wheat plant, and the content of imidacloprid in the roots was higher than that in the leaves. Furthermore, the imidacloprid concentration in the wheat increased with increasing exposure time. After 3 days of exposure, the roots and leaves of the wheat in the 0.5 mg/L treatment group contained 4.55 mg/kg &plusmn; 1.45 mg/kg and 1.30 mg/kg &plusmn; 0.08 mg/kg imidacloprid, respectively, while the roots and leaves of the 5 mg/L treatment group contained 42.5 mg/kg &plusmn; 0.62 mg/kg and 8.71 mg/kg &plusmn; 0.14 mg/kg imidacloprid, respectively. The results from the present study allow for a better understanding of pesticide residues in crops and provide a data reference for the environmental risk assessment of pesticides.

Presented here is a protocol for the determination of the absorption, translocation, and distribution of imidacloprid in wheat under hydroponic conditions using liquid chromatography-tandem mass spectrometry (LC-MS-MS). The results showed that imidacloprid can be absorbed by wheat, and imidacloprid was detected in both the wheat roots and leaves.

Neonicotinoids, a class of insecticides, are widely used because of their novel modes of action, high insecticidal activity, and strong root uptake. ...

Estimating the Integral Metabolite Cytotoxicity of Triazole Pesticides in Plants

Assessing Cytotoxicity of Metabolites of Typical Triazole Pesticides in Plants

Various organic pollutants have been released into the environment because of anthropogenic activities. These pollutants can be taken up by crop plants, causing potential threats to the ecosystem and human health throughout the food chain. The biotransformation of pollutants in plants generates a number of metabolites that may be more toxic than their parent compounds, implying that the metabolites should be taken into account during the toxicity assessment. However, the metabolites of pollutants in plants are extremely complex, making it difficult to comprehensively obtain the toxicological information of all metabolites. This study proposed a strategy to assess the integral cytotoxicity of pollutant metabolites in plants by treating them as a whole during toxicological tests. Triazole pesticides, a class of broad-spectrum fungicides, have been widely applied in agricultural production. Their residue pollution in farmland has drawn increasing attention. Hence, four triazole pesticides, including flusilazole, diniconazole, tebuconazole, and propiconazole, were selected as the tested pollutants. The metabolites were generated by the treatment of carrot callus with tested triazole pesticides. After treatment of 72 h, the metabolites of pesticides in carrot callus were extracted, followed by toxicological tests using the Caco-2 cell line. The results showed that the metabolites of tested pesticides in carrot callus did not significantly inhibit the viability of Caco-2 cells (P>0.05), demonstrating no cytotoxicity of pesticide metabolites. This proposed method opens a new avenue to assess the cytotoxicity of pollutant metabolites in plants, which is expected to provide valuable data for precise toxicity assessment.

Author Spotlight: A New and Efficient Method for Comprehensive Metabolite Cytotoxicity Assessment of Triazole Pesticides in Plants

The protocol describes a new method to assess the integral cytotoxicity of metabolites of triazole pesticides in plants.

Various organic pollutants have been released into the environment because of anthropogenic activities. These pollutants can be taken up by crop plants, ...