Name: a strategy to validate the role of callose-mediated plasmodesmal gating in the tropic response
Uploaded: 2016-04-17T14:00:00
Description: Watch this Scientific Journal Video about A Strategy to Validate the Role of Callose-mediated Plasmodesmal Gating in the Tropic Response at JoVE.com

This research was supported by the National Research Foundation of Korea (NRF-2015R1A2A1A10053576), and by a grant from the Next-Generation BioGreen 21 Program (SSAC, grant PJ01137901), Rural Development Administration, Republic of Korea. RK, WS, ABB and DK were supported by Brain Korea 21 Plus program (BK21+).

1. Screening of Mutants with Altered Phototropic and Gravitropic Responses
<ol>
	<li>Prepare 1x Murashige and Skoog (MS) Medium, pH 5.7, with 0.8% Agar One Day Before the Experiment.
	<ol>
		<li>Add 800 ml of double distilled water to a 2 L conical flask and stir with a magnetic bar.</li>
		<li>Add 4.4 g of MS salt to the conical flask.</li>
		<li>Add 0.5 g of 2-(N-Morpholino) ethane sulphonic acid (MES) and stir until all of the salts are completely dissolved.</li>
		<li>Adjust the pH of the medium to 5.7 with 1 M KOH...

<ol>
	<li>Went, F. W., Thimann, K. V., Bard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phytohormones.">Phytohormones.</a> Experimental biology monographs. , 204 (1937).</li><li>Bennett, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+AUX1+gene:+a+permease-like+regulator+of+root+gravitropism.">Arabidopsis AUX1 gene: a permease-like regulator of root gravitropism.</a> Science. 273 (5277), 948-950 (1996).</li><li>Christensen, S. 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A., Spalding, E. P., Murphy, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+gravi-+and+phototropism+in+plant+mdr+mutants+mislocalizing+the+auxin+efflux+protein+PIN1.">Enhanced gravi- and phototropism in plant mdr mutants mislocalizing the auxin efflux protein PIN1.</a> Nature. 423 (6943), 999-1002 (2003).</li><li>Weijers, D., Friml, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Snap+Shot:+auxin+signaling+and+transport.">Snap Shot: auxin signaling and transport.</a> Cell. 136 (6), 1172-1172 (2009).</li><li>Wisniewska, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polar+PIN+localization+directs+auxin+flow+in+plants.">Polar PIN localization directs auxin flow in plants.</a> Science. 312 (5775), 883 (2006).</li><li>Murphy, A. S., Hoogner, K. R., Peer, W. A., Taiz, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification,+purification,+and+molecular+cloning+of+N-1-naphthylphthalmic+acid-binding+plasma+membrane-associated+aminopeptidases+from+Arabidopsis.">Identification, purification, and molecular cloning of N-1-naphthylphthalmic acid-binding plasma membrane-associated aminopeptidases from Arabidopsis.</a> Plant Physiol. 128 (3), 935-950 (2002).</li><li>Kleine-Vehn, J., Friml, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polar+targeting+and+endocytic+recycling+in+auxin-dependent+plant+development.">Polar targeting and endocytic recycling in auxin-dependent plant development.</a> Annu. Rev. Cell Dev. Biol. 24, 447-473 (2008).</li><li>Yang, Y., Hammes, U. Z., Taylor, C. G., Schachtman, D. P., Nielsen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-affinity+auxin+transport+by+the+AUX1+influx+carrier+protein.">High-affinity auxin transport by the AUX1 influx carrier protein.</a> Curr. Biol. 16 (11), 1123-1127 (2006).</li><li>Geisler, M., Murphy, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ABC+of+auxin+transport:+the+role+of+p-glycoproteins+in+plant+development.">The ABC of auxin transport: the role of p-glycoproteins in plant development.</a> FEBS Lett. 580 (4), 1094-1102 (2006).</li><li>Band, L. 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Sci.USA. 109 (12), 4668-4673 (2012).</li><li>Vanneste, S., Friml, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auxin:+a+trigger+for+change+in+plant+development.">Auxin: a trigger for change in plant development.</a> Cell. 136 (6), 1005-1016 (2009).</li><li>Han, X., 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=Auxin-callose-mediated+plasmodesmal+gating+is+essential+for+tropic+auxin+gradient+formation+and+signaling.">Auxin-callose-mediated plasmodesmal gating is essential for tropic auxin gradient formation and signaling.</a> Dev. Cell. 28 (2), 132-146 (2014).</li><li>Kumar, R., Kumar, D., Hyun, T. K., Kim, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Players+at+plasmodesmal+nano-channels.">Players at plasmodesmal nano-channels.</a> J. Plant Biol. 58, 75-86 (2015).</li><li>Chen, X. Y., 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=The+Arabidopsis+callose+synthase+gene+GSL8+is+required+for+cytokinesis+and+cell+patterning.">The Arabidopsis callose synthase gene GSL8 is required for cytokinesis and cell patterning.</a> Plant Physiol. 150, 105-113 (2009).</li></ol>

In this manuscript, a strategy to screen mutant/over-expression lines that are defective in phototropic and gravitropic responses due to altered PD callose and, hence, PD SEL is described in detail. PD callose synthesis and degradation is mainly accomplished by callose synthases and &#946;-1,3-Glucanases, but regulation of these enzymes is controlled by many upstream factors. To search for such upstream factors or candidates which are directly involved in PD regulation, we have set up this method for screening. This set ...

Plants, as sessile living organisms, have developed a highly sophisticated network of cell-to-cell signaling to address various environmental stimuli. Tropic responses are one of the phenomena by which plants respond to environmental stimuli. Plants show two main tropic responses, phototropism and gravitropism. Photosynthetic plants bend toward the light source by phototropism to harvest maximum energy. Similarly, gravitropism makes the plants to grow toward the gravity center. The fundamental mechanism leading to such tropic responses involves asymmetric gradient formation of the phytohormone auxin1. The act of local auxin gradient formation is well characterized; the genes that are involved in this mechanism provide a roadmap for hormone action2-8. The specific position of auxin efflux carriers, such as PIN-FORMED (PIN) and P-glycoproteins, executes the movement of auxin from the cytoplasm to the cell wall of donor cells9,10. Furthermore, by the active H+/IAA symport activity of auxin influx carriers, such as AUX1/LAX family proteins, auxin is finally delivered to the adjacent receiver cells2,11,12. This directional movement of auxin is known as polar auxin transport (PAT). PAT leads to a differential auxin distribution during various developmental stages and in response to different environmental stimuli13,14. Moreover, the disruption in localization or expression of any of these auxin influx or efflux carriers leads to severe alteration in PAT, which causes a disruption of the auxin gradient, leading to developmental defects. Recently, Han et al. reported that plasmodesmal regulation is also necessary to maintain the auxin gradient15. To date, more than 30 plasmodesmal proteins have been identified16. Among these proteins, AtGSL8 has been reported as a key enzyme for callose synthesis at plasmodesmata (PD) and hence plays a vital role in maintaining the PD size exclusion limit (SEL). Repressed AtGSL8 expression resulted in a distorted auxin gradient pattern leading to no tropic response in contrast to wild type seedlings15.
In this manuscript, methods to explore new candidate genes that are involved in PD regulation are provided. AtGSL8 was used as a model protein to test these methods, as it is a key enzyme contributing to PD callose biosynthesis. Due to the seedling-lethality of gsl8 knock-out mutants17, dexamethasone (dex)-inducible RNAi lines were used in accordance with a previously published report15. The strategy provided here can be adapted to screen genes that are implicated in hypocotyl tropic response controlled by PD SEL.

<table border="0" cellpadding="0" cellspacing="0" width="890">
	<tbody>
		<tr height="45">
			<td height="45" style="height:45px;width:223px;">HPTS (8-Hydroxypyrene -1,3,6-trisulfonic acid trisodium salt)</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:175px;">H1529-1G</td>
			<td style="width:335px;">Fluorescent dye as symplasmic tracer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">LE Agarose</td>
			<td style="width:157px;">Dongin-Genomic</td>
			<td style="width:175px;">GEL001-500G</td>
			<td style="width:335px;">Used for HPTS agarose block</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Microwave oven</td>
			<td style="width:157px;">LG-Goldstar</td>
			<td style="width:175px;"></td>
			<td style="width:335px;">Machine for boiling agarose gel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">100 mL glass conical flask</td>
			<td style="width:157px;">Dong Kwang</td>
			<td style="width:175px;">A0205</td>
			<td style="width:335px;">Used to boil HPTS agarose gel</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:223px;">Petri Dish (35x10 mm)</td>
			<td style="width:157px;">SPL life sciences</td>
			<td style="width:175px;">SPL10035</td>
			<td style="width:335px;">Used to make HPTS agarose blocks and wash plant samples&#160;</td>
		</tr>
		<tr height="79">
			<td height="79" style="height:79px;width:223px;">Microscope cover slides and glass slides (24 x 50 mm)</td>
			<td style="width:157px;">Marienfeld Laboratory Glassware</td>
			<td style="width:175px;">101222</td>
			<td style="width:335px;">Used for HPTS agarose blocks and microscopic sample preparation</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:223px;">MS medium plates 125 x 125 x 20 mm</td>
			<td style="width:157px;">SPL life sciences</td>
			<td style="width:175px;">SPL11125</td>
			<td style="width:335px;">Plates to make MS agar medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Scissors</td>
			<td style="width:157px;">Germany Stainless</td>
			<td style="width:175px;">HSB 942-11</td>
			<td style="width:335px;">Used to excise hook region of plant samples</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:223px;">Murashige and Skoog (MS) basal salt mixture</td>
			<td style="width:157px;">Duchefa</td>
			<td style="width:175px;">P10453.01</td>
			<td style="width:335px;">MS medium including vitamins.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">(N-morpholino) ethanesulfonic acid (MES) monohydrate</td>
			<td style="width:157px;">Bioshop</td>
			<td style="width:175px;">3G30212</td>
			<td style="width:335px;">To make MS media.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Plant agar</td>
			<td style="width:157px;">Duchefa</td>
			<td style="width:175px;">P1001.1000</td>
			<td style="width:335px;">To solidify MS media.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Autoclave</td>
			<td style="width:157px;">ALP</td>
			<td style="width:175px;">CL-40L</td>
			<td style="width:335px;"></td>
		</tr>
		<tr height="56">
			<td height="56" style="height:56px;width:223px;">Shaker</td>
			<td style="width:157px;">Wise Mix</td>
			<td style="width:175px;">SHO-1D</td>
			<td style="width:335px;">To wash off the aniline blue staining buffer and HPTS dye in a placid way.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">1 ml Blue tips</td>
			<td style="width:157px;">Sorenson</td>
			<td style="width:175px;">10040</td>
			<td style="width:335px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">1 ml pipette</td>
			<td style="width:157px;">BioPette</td>
			<td style="width:175px;">L-1101-2</td>
			<td style="width:335px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Surgical tape</td>
			<td style="width:157px;">MIcropore</td>
			<td style="width:175px;">1530-0</td>
			<td style="width:335px;">To seal the MS plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Aniline blue (Methyl blue)</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:175px;">M5528-25G</td>
			<td style="width:335px;">Used to prepare aniline blue staining buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Glycine</td>
			<td style="width:157px;">Bioshop</td>
			<td style="width:175px;">GLN001</td>
			<td style="width:335px;">Used to prepare aniline blue staining buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">DDG</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:175px;">D8375-1G</td>
			<td style="width:335px;">Used for the inhibition of callose synthases.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:223px;">Confocal microscope</td>
			<td style="width:157px;">Olympus</td>
			<td style="width:175px;">FV1000MPE SIM</td>
			<td style="width:335px;">To check aniline blue staining and HPTS dye loading result.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Stirrer&#160;</td>
			<td style="width:157px;">I lab&#160;</td>
			<td style="width:175px;">K400</td>
			<td style="width:335px;">To mix media solution.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Aluminium foil</td>
			<td style="width:157px;">SW cooking foil</td>
			<td style="width:175px;"></td>
			<td style="width:335px;">To wrap plates in a dark condition.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Sodium hypochlorite</td>
			<td style="width:157px;">Samjin Industry</td>
			<td style="width:175px;"></td>
			<td style="width:335px;">To surface-sterilized seeds</td>
		</tr>
	</tbody>
</table>

a strategy to validate the role of callose-mediated plasmodesmal gating in the tropic response

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The establishment of an auxin gradient is a key event leading to phototropism and gravitropism. Previously, polar auxin transport (PAT) was shown to establish an auxin gradient in different cellular domains of plants. However, Han et al. recently demonstrated that for proper auxin gradient formation, plasmodesmal callose-mediated symplasmic connectivity between the adjacent cells is also a critical factor. In this manuscript, the strategy to elucidate the role of particular genes, which can affect phototropism and gravitropism by altering the symplasmic connectivity through modulating plasmodesmal callose synthesis, is discussed. The first step is to screen aberrant tropic responses from 3-day-old etiolated seedlings of mutants or over-expression lines of a gene along with the wild type. This preliminary screening can lead to the identification of a range of genes functioning in PAT or controlling symplasmic connectivity. The second screening involves the sorting of candidates that show altered tropic responses by affecting symplasmic connectivity. To address such candidates, the movement of a symplasmic tracer and the deposition of plasmodesmal callose were examined. This strategy would be useful to explore new candidate genes that can regulate symplasmic connectivity directly or indirectly during tropic responses and other developmental processes.

In the current setup, dexamethasone (dex)-inducible RNAi lines of AtGSL8 [hereafter dsGSL8 (+dex/-dex)] were used, as homozygous gsl8 T-DNA insertion mutants are seedling lethal18. Three-day-old etiolated seedlings of dsGSL8 and wild type seedlings with &#177;dex were exposed to phototropic and gravitropic stimuli. We found that dsGSL8 (+dex) seedlings were defective in phototropism and gravitropism15. Figure 5</st...

Watch this Scientific Journal Video about A Strategy to Validate the Role of Callose-mediated Plasmodesmal Gating in the Tropic Response at JoVE.com

A Strategy to Validate the Role of Callose-mediated Plasmodesmal Gating in the Tropic Response

This article describes the methods for screening the genes controlling plasmodesmal permeability and hence auxin gradient during tropic response. This includes the measurement of the degree of tropic response in hypocotyl of Arabidopsis thaliana and checking plasmodesmal permeability by 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) loading and finally callose level assessment.

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The ...

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