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

Denne forskning blev støttet af Grundforskningsfonden Korea (NRF-2015R1A2A1A10053576), og af en bevilling fra den næste generation af Biogreen 21 Program (SSAC yde PJ01137901), Udvikling af Landdistrikterne Administration, Republikken Korea. RK, WS, ABB og DK blev støttet af Brain Sydkorea 21 Plus-programmet (BK21 +).

1. Screening af mutanter med ændret fototropisk og Gravitropic Responses <ol><li> Forbered 1x Murashige og Skoog (MS) Medium, pH 5,7, med 0,8% Agar en dag før forsøget. <ol><li> Tilsættes 800 ml dobbelt destilleret vand til en 2 L konisk kolbe og omrøres med en magnetisk stang. </li><li> Tilføj 4,4 g MS salt til den koniske kolbe. </li><li> Tilsæt 0,5 g 2- (N-morpholino) ethansulfonsyre (MES) og omrør indtil alle saltene er fuldstændig opløst. </li><li> Justere mediets pH til 5,7 med 1 M KOH. </li><li> Overf...

<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. 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=Root+gravitropism+is+regulated+by+a+transient+lateral+auxin+gradient+controlled+by+a+tipping+point+mechanism.">Root gravitropism is regulated by a transient lateral auxin gradient controlled by a tipping point mechanism.</a> Proc. Natl. Acad. 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>

I dette manuskript, til en strategi screene mutant / over-udtryk linjer, der er defekte fototropisk og gravitropic reaktioner som følge af ændret PD callose og dermed er PD SEL beskrevet i detaljer. PD callose syntese og nedbrydning hovedsageligt opnås ved callose syntaser og β-1,3-glucanaser, men reguleringen af ​​disse enzymer er kontrolleret af mange opstrøms faktorer. For at søge efter sådanne opstrøms faktorer eller kandidater, der er direkte involveret i PD regulering, har vi oprettet denne metode til ...

Planter, som sessile levende organismer, har udviklet en meget avanceret netværk af celle-til-cellesignalering at løse forskellige miljømæssige stimuli. Tropic responser er en af ​​de fænomener, som planter reagerer på miljømæssige stimuli. Planter viser to vigtigste tropiske svar, fototropisme og gravitropism. Fotosyntetiske planter bøje mod lyskilden ved fototropisme at høste maksimal energi. Tilsvarende gravitropism gør planterne at vokse mod tyngdepunkt. Den grundlæggende mekanisme, der fører til sådanne tropiske reaktioner involverer asymmetrisk gradient dannelse af phytohormonet auxin en. Det handler om lokale auxin gradient dannelse er godt karakteriseret; de gener, der er involveret i denne mekanisme giver en køreplan for hormon handling 2-8. Den specifikke placering af auxin efflux bærere, såsom PIN-formed (PIN) og p-glycoproteiner, udfører bevægelsen af auxin fra cytoplasmaet til cellevæggen af donorceller 9,10. Furthermore, ved den aktive H + / IAA symport aktivitet af auxin tilstrømning bærere, såsom AUX1 / LAX familie proteiner, auxin er endelig leveret til de tilstødende modtager celler 2,11,12. Denne retningsbestemt bevægelse af auxin er kendt som polar auxin transport (PAT). PAT fører til en differentiel auxin fordeling under forskellige udviklingsstadier og som reaktion på forskellige miljømæssige stimuli 13,14. Desuden afbrydelse i lokalisering eller ekspression af nogen af ​​disse auxin indstrømning eller efflux bærere fører til alvorlig ændring i PAT, som forårsager en afbrydelse af den auxin-gradient, hvilket fører til udviklingsmæssige defekter. For nylig, Han et al. rapporterede, at plasmodesmal forordning er også nødvendig for at opretholde auxin gradient 15. Til dato har mere end 30 plasmodesmal proteiner blevet identificeret 16. Blandt disse proteiner har AtGSL8 blevet rapporteret som et centralt enzym til callose syntese ved plasmodesmata (PD), og derfor spiller en afgørende rolle i maintaining grænse PD udelukkelse størrelse (SEL). Undertrykt AtGSL8 udtryk resulterede i en forvrænget auxin gradient mønster fører til ingen tropiske respons i modsætning til vildtype kimplanter 15. I dette manuskript, metoder udforske nye kandidatgener, der er involveret i PD regulering leveres. AtGSL8 blev anvendt som en model protein for at teste disse metoder, da det er et vigtigt enzym bidrager til PD callose biosyntese. På grund af den kimplante-letalitet gsl8 knock-out mutanter 17, blev dexamethason (dex) inducerbare RNAi linjer anvendes i overensstemmelse med en tidligere offentliggjort rapport 15. Oplysningerne her strategi kan tilpasses til at screene gener, der er impliceret i hypokotyle tropic respons kontrolleret af 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

Anlægget hormon auxin spiller en vigtig rolle i mange vækst- og udviklingsprocesser, herunder tropiske reaktioner på lys og tyngdekraft. Etableringen af ​​en auxin gradient er en central begivenhed, der medfører fototropisme og gravitropism. Tidligere blev polar auxin transport (PAT) vist at etablere en auxin gradient i forskellige cellulære domæner af planter. Men Han et al. Nylig vist, at for korrekt auxin gradient dannelse, plasmodesmal callose-medieret symplasmic konnektivitet mellem de tilstødende celler er også en kritisk faktor. I dette manuskript, til den strategi, belyse den rolle, bestemte gener, som kan påvirke fototropisme og gravitropism ved at ændre symplasmic tilslutning via modulerende plasmodesmal callose syntese, diskuteres. Det første skridt er at screene afvigende tropiske svar fra 3 dage gamle etiolerede frøplanter af mutanter eller over-udtryk linjer af et gen sammen med den vilde type. Denne indledende screeningkan føre til identifikation af en række gener fungerer i PAT eller kontrollerende symplasmic tilslutningsmuligheder. Den anden screening indebærer sortering af kandidater, der viser ændrede tropiske svar ved at påvirke symplasmic tilslutningsmuligheder. For at imødegå sådanne kandidater, blev bevægelsen af ​​en symplasmic sporstof og aflejring af plasmodesmal callose undersøgt. Denne strategi vil være nyttigt at udforske nye kandidatgener, der kan regulere symplasmic konnektivitet direkte eller indirekte under tropic svar og andre udviklingsprocesser.

I den nuværende setup, dexamethason (dex) inducerbare RNAi linjer af AtGSL8 [herefter dsGSL8 (+ k s / -dex)] blev anvendt, som homozygot gsl8 T-DNA indsættelse mutanter er sætteplante dødbringende 18. Tre dage gamle etiolerede frøplanter af dsGSL8 og vild type frøplanter med ± dex blev udsat for fototropisk og gravitropic stimuli. Vi fandt, at dsGSL8 (+ dex) kimplanter var defekte i fototropisme og gravitropism 15....

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

Denne artikel beskriver metoderne til screening af gener, der kontrollerer plasmodesmal permeabilitet og dermed auxin gradient under tropiske respons. Dette omfatter måling af graden af ​​tropiske respons i hypokotyle af Arabidopsis thaliana og kontrol plasmodesmal permeabilitet med 8-hydroxypyren-1,3,6-trisulfonsyre (HPTS) lastning og vurdering endelig callose niveau.

En strategi at validere rolle Callose-medieret Plasmodesmal gating i Tropic svar

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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Kombinere mitotiske celle synkronisering og høj opløsning Konfokal mikroskopi for at studere rollen af multifunktionelle cellecyklus proteiner under mitosen

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...

Isolation and 3D Collagen Sandwich Culture of Primary Mouse Hepatocytes to Study the Role of Cytoskeleton in Bile Canalicular Formation In Vitro

Isolation og 3D kollagen sandwich kultur af primære mus hepatocytter at studere rollen af cytoskelet i galde Canalicular dannelse in vitro

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or ...