BACKGROUND: Processes of anterograde and retrograde membrane trafficking play an important role in cellular homeostasis and dynamic rearrangements of the plasma membrane (PM) in all eukaryotes. These processes depend on the activity of adenosine ribosylation factors (ARFs), a family of GTP-binding proteins and their guanine exchange factors (GEFs). However, knowledge on the function and specificity of individual ARF-GEFs for individual steps of membrane trafficking pathways is still limited in plants. RESULTS: In this work, treatments with various trafficking inhibitors showed that the endocytosis of FM 4-64 is largely dynamin-dependent and relies on proteins containing endocytic tyrosine-based internalization motif and intact cytoskeleton. Interestingly, brefeldin A (BFA), reported previously as an inhibitor of anterograde membrane trafficking in plants, appeared to be the most potent inhibitor of endocytosis in tobacco. In concert with this finding, we demonstrate that the point mutation in the Sec7 domain of the GNOM-LIKE protein1a (NtGNL1a) confers intracellular trafficking pathway-specific BFA resistance. The internalization of FM 4-64 and trafficking of PIN-FORMED1 (PIN1) auxin efflux carrier in BY-2 tobacco cells were studied to reveal the function of the ARF-GEF NtGNL1a in these. CONCLUSIONS: Altogether, our observations uncovered the role of NtGNL1a in endocytosis, including endocytosis of PM proteins (as PIN1 auxin efflux carrier). Moreover these data emphasize the need of careful evaluation of mode of action of non-native inhibitors in various species. In addition, they demonstrate the potential of tobacco BY-2 cells for selective mapping of ARF-GEF-regulated endomembrane trafficking pathways.

Department of Experimental Plant Biology Faculty of Science Charles University Prague Viničná 5 128 44 Prague 2 Czech Republic

Institute of Experimental Botany Academy of Sciences of the Czech Republic Rozvojová 263 165 02 Prague 6 Czech Republic

See more in PubMed

Lam S, Siu C, Hillmer S, Jang S, An G, Robinson D, et al. Rice SCAMP1 defines clathrin-coated, trans-golgi-located tubular-vesicular structures as an early endosome in tobacco BY-2 cells. Plant Cell. 2007;19:296–319. doi: 10.1105/tpc.106.045708. PubMed DOI PMC

Robinson D, Jiang L, Schumacher K. The endosomal system of plants: charting new and familiar territories. Plant Physiol. 2008;147:1482–1492. doi: 10.1104/pp.108.120105. PubMed DOI PMC

Hwang I, Robinson D. Transport vesicle formation in plant cells. Curr Opin Plant Biol. 2009;12:660–669. doi: 10.1016/j.pbi.2009.09.012. PubMed DOI

Yorimitsu T, Sato K, Takeuchi M. Molecular mechanisms of Sar/Arf GTPases in vesicular trafficking in yeast and plants. Front Plant Sci. 2014;5:411. doi: 10.3389/fpls.2014.00411. PubMed DOI PMC

Ito E, Fujimoto M, Ebine K, Uemura T, Ueda T, Nakano A. Dynamic behavior of clathrin in Arabidopsis thaliana unveiled by live imaging. Plant J. 2012;69:204–216. doi: 10.1111/j.1365-313X.2011.04782.x. PubMed DOI

Dhonukshe P, Aniento F, Hwang I, Robinson D, Mravec J, Stierhof Y, et al. Clathrin-mediated constitutive endocytosis of PIN auxin efflux carriers in Arabidopsis. Curr Biol. 2007;17:520–527. doi: 10.1016/j.cub.2007.01.052. PubMed DOI

Geldner N, Jürgens G. Endocytosis in signalling and development. Curr Opin Plant Biol. 2006;9:589–594. doi: 10.1016/j.pbi.2006.09.011. PubMed DOI

Pérez-Gómez J, Moore I. Plant endocytosis: it is clathrin after all. Curr Biol. 2007;17:R217–219. doi: 10.1016/j.cub.2007.01.045. PubMed DOI

Barberon M, Zelazny E, Robert S, Conéjéro G, Curie C, Friml J, et al. Monoubiquitin-dependent endocytosis of the iron-regulated transporter 1 (IRT1) transporter controls iron uptake in plants. Proc Natl Acad Sci U S A. 2011;108:E450–458. doi: 10.1073/pnas.1100659108. PubMed DOI PMC

Di Rubbo S, Irani NG, Kim SY, Xu ZY, Gadeyne A, Dejonghe W, et al. The clathrin adaptor complex AP-2 mediates endocytosis of brassinosteroid insensitive1 in Arabidopsis. Plant Cell. 2013;25:2986–2997. doi: 10.1105/tpc.113.114058. PubMed DOI PMC

Irani NG, Di Rubbo S, Mylle E, Van den Begin J, Schneider-Pizoń J, Hniliková J, et al. Fluorescent castasterone reveals BRI1 signaling from the plasma membrane. Nat Chem Biol. 2012;8:583–589. doi: 10.1038/nchembio.958. PubMed DOI

Takano J, Tanaka M, Toyoda A, Miwa K, Kasai K, Fuji K, et al. Polar localization and degradation of Arabidopsis boron transporters through distinct trafficking pathways. Proc Natl Acad Sci U S A. 2010;107:5220–5225. doi: 10.1073/pnas.0910744107. PubMed DOI PMC

Geldner N, Anders N, Wolters H, Keicher J, Kornberger W, Muller P, et al. The Arabidopsis GNOM ARF-GEF mediates endosomal recycling, auxin transport, and auxin-dependent plant growth. Cell. 2003;112:219–230. doi: 10.1016/S0092-8674(03)00003-5. PubMed DOI

Naramoto S, Kleine-Vehn J, Robert S, Fujimoto M, Dainobu T, Paciorek T, et al. ADP-ribosylation factor machinery mediates endocytosis in plant cells. Proc Natl Acad Sci U S A. 2010;107:21890–21895. doi: 10.1073/pnas.1016260107. PubMed DOI PMC

Teh OK, Moore I. An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells. Nature. 2007;448:493–496. doi: 10.1038/nature06023. PubMed DOI

Hay J, Scheller R. SNAREs and NSF in targeted membrane fusion. Curr Opin Cell Biol. 1997;9:505–512. doi: 10.1016/S0955-0674(97)80026-9. PubMed DOI

Sanderfoot A, Raikhel N. The specificity of vesicle trafficking: coat proteins and SNAREs. Plant Cell. 1999;11:629–642. doi: 10.1105/tpc.11.4.629. PubMed DOI PMC

Eliáš M, Drdová E, Ziak D, Bavlnka B, Hala M, Cvrčkova F, et al. The exocyst complex in plants. Cell Biol Int. 2003;27:199–201. doi: 10.1016/S1065-6995(02)00349-9. PubMed DOI

Fendrych M, Synek L, Pečenková T, Drdová EJ, Sekeres J, de Rycke R, et al. Visualization of the exocyst complex dynamics at the plasma membrane of Arabidopsis thaliana. Mol Biol Cell. 2013;24:510–520. doi: 10.1091/mbc.E12-06-0492. PubMed DOI PMC

Synek L, Schlager N, Eliáš M, Quentin M, Hauser M, Žárský V. AtEXO70A1, a member of a family of putative exocyst subunits specifically expanded in land plants, is important for polar growth and plant development. Plant J. 2006;48:54–72. doi: 10.1111/j.1365-313X.2006.02854.x. PubMed DOI PMC

Barker SA, Caldwell KK, Hall A, Martinez AM, Pfeiffer JR, Oliver JM, et al. Wortmannin blocks lipid and protein kinase activities associated with PI 3-kinase and inhibits a subset of responses induced by Fc epsilon R1 cross-linking. Mol Biol Cell. 1995;6:1145–1158. doi: 10.1091/mbc.6.9.1145. PubMed DOI PMC

Matsuoka K, Higuchi T, Maeshima M, Nakamura K. A Vacuolar-Type H + −ATPase in a Nonvacuolar Organelle Is Required for the Sorting of Soluble Vacuolar Protein Precursors in Tobacco Cells. Plant Cell. 1997;9:533–546. PubMed PMC

Grebe M, Xu J, Möbius W, Ueda T, Nakano A, Geuze H, et al. Arabidopsis sterol endocytosis involves actin-mediated trafficking via ARA6-positive early endosomes. Curr Biol. 2003;13:1378–1387. doi: 10.1016/S0960-9822(03)00538-4. PubMed DOI

Miller RG. The use and abuse of filipin to localize cholesterol in membranes. Cell Biol Int Rep. 1984;8:519–535. doi: 10.1016/0309-1651(84)90050-X. PubMed DOI

Macia E, Ehrlich M, Massol R, Boucrot E, Brunner C, Kirchhausen T. Dynasore, a cell-permeable inhibitor of dynamin. Dev Cell. 2006;10:839–50. PubMed

Newton A, Kirchhausen T, Murthy V. Inhibition of dynamin completely blocks compensatory synaptic vesicle endocytosis. Proc Natl Acad Sci U S A. 2006;103:17955–17960. doi: 10.1073/pnas.0606212103. PubMed DOI PMC

Paciorek T, Zažímalová E, Ruthardt N, Petrášek J, Stierhof Y, Kleine-Vehn J, et al. Auxin inhibits endocytosis and promotes its own efflux from cells. Nature. 2005;435:1251–1256. doi: 10.1038/nature03633. PubMed DOI

Baluška F, Hlavacka A, Šamaj J, Palme K, Robinson DG, Matoh T, et al. F-actin-dependent endocytosis of cell wall pectins in meristematic root cells. Insights from brefeldin a-induced compartments. Plant Physiol. 2002;130(1):422–431. doi: 10.1104/pp.007526. PubMed DOI PMC

Geldner N, Friml J, Stierhof Y, Jürgens G, Palme K. Auxin transport inhibitors block PIN1 cycling and vesicle trafficking. Nature. 2001;413:425–428. doi: 10.1038/35096571. PubMed DOI

Lisboa S, Scherer G, Quader H. Localized endocytosis in tobacco pollen tubes: visualisation and dynamics of membrane retrieval by a fluorescent phospholipid. Plant Cell Rep. 2008;27:21–28. doi: 10.1007/s00299-007-0437-1. PubMed DOI

Ovečka M, Lang I, Baluška F, Ismail A, Illes P, Lichtscheidl IK. Endocytosis and vesicle trafficking during tip growth of root hairs. Protoplasma. 2005;226:39–54. doi: 10.1007/s00709-005-0103-9. PubMed DOI

Šamaj J, Baluška F, Voigt B, Schlicht M, Volkmann D, Menzel D. Šamaj Endocytosis, Actin Cytoskeleton, and Signaling. Plant Physiol. 2004;135(3):1150–1161. doi: 10.1104/pp.104.040683. PubMed DOI PMC

Takáč T, Pechan T, Richter H, Müller J, Eck C, Böhm N, et al. Proteomics on brefeldin a-treated Arabidopsis roots reveals profilin 2 as a new protein involved in the cross-talk between vesicular trafficking and the actin cytoskeleton. J Proteome Res. 2011;10(2):488–501. doi: 10.1021/pr100690f. PubMed DOI

Boutté Y, Crosnier M, Carraro N, Traas J, Satiat-Jeunemaitre B. The plasma membrane recycling pathway and cell polarity in plants: studies on PIN proteins. J Cell Sci. 2006;119:1255–1265. doi: 10.1242/jcs.02847. PubMed DOI

Jackson C, Casanova J. Turning on ARF: the Sec7 family of guanine-nucleotide-exchange factors. Trends Cell Biol. 2000;10:60–67. doi: 10.1016/S0962-8924(99)01699-2. PubMed DOI

Dhonukshe P, Baluska F, Schlicht M, Hlavacka A, Samaj J, Friml J, et al. Endocytosis of cell surface material mediates cell plate formation during plant cytokinesis. Dev Cell. 2006;10:137–150. doi: 10.1016/j.devcel.2005.11.015. PubMed DOI

Malínská K, Jelínková A, Petrášek J. The use of FM dyes to analyze plant endocytosis. Methods Mol Biol. 2014;1209:1–11. doi: 10.1007/978-1-4939-1420-3_1. PubMed DOI

Banbury D, Oakley J, Sessions R, Banting G. Tyrphostin A23 inhibits internalization of the transferrin receptor by perturbing the interaction between tyrosine motifs and the medium chain subunit of the AP-2 adaptor complex. J Biol Chem. 2003;278:12022–12028. doi: 10.1074/jbc.M211966200. PubMed DOI

Ortiz-Zapater E, Soriano-Ortega E, Marcote M, Ortiz-Masiá D, Aniento F. Trafficking of the human transferrin receptor in plant cells: effects of tyrphostin A23 and brefeldin A. Plant J. 2006;48:757–770. doi: 10.1111/j.1365-313X.2006.02909.x. PubMed DOI

Lam S, Cai Y, Tse Y, Wang J, Law A, Pimpl P, et al. BFA-induced compartments from the Golgi apparatus and trans-Golgi network/early endosome are distinct in plant cells. Plant J. 2009;60:865–881. doi: 10.1111/j.1365-313X.2009.04007.x. PubMed DOI

Emans N, Zimmermann S, Fischer R. Uptake of a fluorescent marker in plant cells is sensitive to brefeldin A and wortmannin. Plant Cell. 2002;14:71–86. doi: 10.1105/tpc.010339. PubMed DOI PMC

Wang J, Cai Y, Miao Y, Lam SK, Jiang L. Wortmannin induces homotypic fusion of plant prevacuolar compartments. J Exp Bot. 2009;60:3075–3083. doi: 10.1093/jxb/erp136. PubMed DOI PMC

Aniento F, Robinson D. Testing for endocytosis in plants. Protoplasma. 2005;226:3–11. doi: 10.1007/s00709-005-0101-y. PubMed DOI

Kleine-Vehn J, Dhonukshe P, Swarup R, Bennett M, Friml J. Subcellular trafficking of the Arabidopsis auxin influx carrier AUX1 uses a novel pathway distinct from PIN1. Plant Cell. 2006;18:3171–3181. doi: 10.1105/tpc.106.042770. PubMed DOI PMC

Sharfman M, Bar M, Ehrlich M, Schuster S, Melech-Bonfil S, Ezer R, et al. Endosomal signaling of the tomato leucine-rich repeat receptor-like protein LeEix2. Plant J. 2011;68:413–423. doi: 10.1111/j.1365-313X.2011.04696.x. PubMed DOI

Fan L, Li R, Pan J, Ding Z, Lin J. Endocytosis and its regulation in plants. Trends Plant Sci. 2015;20:388–397. doi: 10.1016/j.tplants.2015.03.014. PubMed DOI

Wang Q, Kong L, Hao H, Wang X, Lin J, Šamaj J, et al. Effects of Brefeldin A on Pollen Germination and Tube Growth. Antagonistic Effects on Endocytosis and Secretion. Plant Phys. 2005;139:1692–1703. doi: 10.1104/pp.105.069765. PubMed DOI PMC

Jelínková A, Malínská K, Simon S, Kleine-Vehn J, Pařezová M, Pejchar P, et al. Probing plant membranes with FM dyes: tracking, dragging or blocking? Plant J. 2010;61:883–892. doi: 10.1111/j.1365-313X.2009.04102.x. PubMed DOI

Ritzenthaler C, Nebenführ A, Movafeghi A, Stussi-Garaud C, Behnia L, Pimpl P, et al. Reevaluation of the effects of brefeldin A on plant cells using tobacco Bright Yellow 2 cells expressing Golgi-targeted green fluorescent protein and COPI antisera. Plant Cell. 2002;14:237–261. doi: 10.1105/tpc.010237. PubMed DOI PMC

Satiat-Jeunemaitre B. Spatial and temporal regulations in helicoidal extracellular matrices: comparison between plant and animal systems. Tissue Cell. 1992;24:315–334. doi: 10.1016/0040-8166(92)90049-D. PubMed DOI

Tse Y, Mo B, Hillmer S, Zhao M, Lo S, Robinson D, et al. Identification of multivesicular bodies as prevacuolar compartments in Nicotiana tabacum BY-2 cells. Plant Cell. 2004;16:672–693. doi: 10.1105/tpc.019703. PubMed DOI PMC

Hess M, Müller M, Debbage P, Vetterlein M, Pavelka M. Cryopreparation provides new insight into the effects of brefeldin A on the structure of the HepG2 Golgi apparatus. J Struct Biol. 2000;130:63–72. doi: 10.1006/jsbi.2000.4230. PubMed DOI

Petrášek J, Černá A, Schwarzerová K, Elčkner M, Morris D, Zažímalová E. Do phytotropins inhibit auxin efflux by impairing vesicle traffic? Plant Physiol. 2003;131:254–263. doi: 10.1104/pp.012740. PubMed DOI PMC

Matsuoka K, Bassham D, Raikhel N, Nakamura K. Different sensitivity to wortmannin of two vacuolar sorting signals indicates the presence of distinct sorting machineries in tobacco cells. J Cell Biol. 1995;130:1307–1318. doi: 10.1083/jcb.130.6.1307. PubMed DOI PMC

Jaillais Y, Fobis-Loisy I, Miège C, Rollin C, Gaude T. AtSNX1 defines an endosome for auxin-carrier trafficking in Arabidopsis. Nature. 2006;443:106–9. PubMed

Petrášek J, Mravec J, Bouchard R, Blakeslee J, Abas M, Seifertová D, et al. PIN proteins perform a rate-limiting function in cellular auxin efflux. Science. 2006;312:914–918. doi: 10.1126/science.1123542. PubMed DOI

Kleine-Vehn J, Dhonukshe P, Sauer M, Brewer P, Wiśniewska J, Paciorek T, et al. ARF GEF-dependent transcytosis and polar delivery of PIN auxin carriers in Arabidopsis. Curr Biol. (2008a-a) 18, 526–531. PubMed

Kleine-Vehn J, Langowski L, Wisniewska J, Dhonukshe P, Brewer P and Friml J. Cellular and molecular requirements for polar PIN targeting and transcytosis in plants. Mol Plant. (2008a-b) 1, 1056–1066. PubMed

Sata M, Donaldson JG, Moss J, Vaughan M. Brefeldin A-inhibited guanine nucleotide-exchange activity of Sec7 domain from yeast Sec7 with yeast and mammalian ADP ribosylation factors. Proc Natl Acad Sci U S A. 1998;95:4204–4208. doi: 10.1073/pnas.95.8.4204. PubMed DOI PMC

Wang L, Liao FL, Zhu L, Peng XB, Sun MX. NtGNL1 is involved in embryonic cell division patterning, root elongation, and pollen tube growth in tobacco. New Phytol. 2008;179:81–93. doi: 10.1111/j.1469-8137.2008.02444.x. PubMed DOI

Liao F, Wang L, Yang LB, Peng X, Sun M. NtGNL1 plays an essential role in pollen tube tip growth and orientation likely via regulation of post-Golgi trafficking. PLoS One. 2010;5:e13401. doi: 10.1371/journal.pone.0013401. PubMed DOI PMC

Sierro N, van Oeveren J, van Eijk MJ, Martin F, Stormo KE, Peitsch MC, et al. Whole genome profiling physical map and ancestral annotation of tobacco Hicks Broadleaf. Plant J. 2013;75:880–889. doi: 10.1111/tpj.12247. PubMed DOI PMC

Geldner N, Richter S, Vieten A, Marquardt S, Torres-Ruiz R, Mayer U, et al. Partial loss-of-function alleles reveal a role for GNOM in auxin transport-related, post-embryonic development of Arabidopsis. Development. 2004;131:389–400. doi: 10.1242/dev.00926. PubMed DOI

Naramoto S, Otegui MS, Kutsuna N, de Rycke R, Dainobu T, Karampelias M, et al. Insights into the localization and function of the membrane trafficking regulator GNOM ARF-GEF at the Golgi apparatus in Arabidopsis. Plant Cell. 2014;26:3062–3076. doi: 10.1105/tpc.114.125880. PubMed DOI PMC

Richter S, Geldner N, Schrader J, Wolters H, Stierhof Y, Rios G, et al. Functional diversification of closely related ARF-GEFs in protein secretion and recycling. Nature. 2007;448:488–492. doi: 10.1038/nature05967. PubMed DOI

Richter S, Müller LM, Stierhof YD, Mayer U, Takada N, Kost B, et al. Polarized cell growth in Arabidopsis requires endosomal recycling mediated by GBF1-related ARF exchange factors. Nat Cell Biol. 2012;14:80–86. doi: 10.1038/ncb2389. PubMed DOI

Richter S, Kientz M, Brumm S, Nielsen ME, Park M, Gavidia R, et al. Delivery of endocytosed proteins to the cell-division plane requires change of pathway from recycling to secretion. eLife. 2014;3:e02131. doi: 10.7554/eLife.02131. PubMed DOI PMC

Tanaka H, Kitakura S, De Rycke R, De Groodt R, Friml J. Fluorescence imaging-based screen identifies ARF GEF component of early endosomal trafficking. Curr Biol. 2009;19:391–397. doi: 10.1016/j.cub.2009.01.057. PubMed DOI

Tanaka H, Kitakura S, Rakusová H, Uemura T, Feraru MI, De Rycke R, et al. Cell polarity and patterning by PIN trafficking through early endosomal compartments in Arabidopsis thaliana. PLoS Genet. 2013;9:e1003540. doi: 10.1371/journal.pgen.1003540. PubMed DOI PMC

Anders N, Nielsen M, Keicher J, Stierhof YD, Furutani M, Tasaka M, et al. Membrane association of the Arabidopsis ARF exchange factor GNOM involves interaction of conserved domains. Plant Cell. 2008;20:142–151. doi: 10.1105/tpc.107.056515. PubMed DOI PMC

Bandmann V, Kreft M, Homann U. Modes of Exocytotic and Endocytotic Events in Tobacco BY-2 Protoplasts. Molecular Plant. 2011;4:241–251. doi: 10.1093/mp/ssq072. PubMed DOI

Nagata T, Nemoto Y, Hasezawa S. Tobacco BY-2 cell line as the "HeLa" cell in the cell biology of higher plants. Int Rev Cytol. 1992;130:1–30. doi: 10.1016/S0074-7696(08)62452-3. DOI

Benková E, Michniewicz M, Sauer M, Teichmann T, Seifertová D, Jürgens G, et al. Local, efflux-dependent auxin gradients as a common module for plant organ formation. Cell. 2003;115:591–602. doi: 10.1016/S0092-8674(03)00924-3. PubMed DOI

Zažímalová E, Krecek P, Skůpa P, Hoyerová K, Petrásek J. Polar transport of the plant hormone auxin - the role of PIN-FORMED (PIN) proteins. Cell Mol Life Sci. 2007;64:1621–1637. doi: 10.1007/s00018-007-6566-4. PubMed DOI PMC

An G. High efficiency transformation of cultured tobacco cells. Plant Physiol. 1985;79:568–570. doi: 10.1104/pp.79.2.568. PubMed DOI PMC

Zuo J, Niu QW, Chua NH. Technical advance: An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants. Plant J. 2000;24:265–273. doi: 10.1046/j.1365-313x.2000.00868.x. PubMed DOI

Opatrný Z, Opatrná J. The specificity of the effect of 2,4-D and NAA on the growth, micromorphology, and occurrence of starch in long-term Nicotiana tabacum L. cell strains. Biol Plant. 1976;18:359–365. doi: 10.1007/BF02922462. DOI

Axelos M, Curie C, Mazzolini L, Bardet C, Lescure B. A protocol for transient gene expression in Arabidopsis thaliana protoplasts isolated from cell suspension cultures. Plant Physiol Biochem. 1992;30:123–128.

Heller, R. (1953) Studies on the mineral nutrition of in vitro plant tissue cultures. Ann Sci Nat Bot Biol Veg, pp. 1–223.

No Time for Transcription-Rapid Auxin Responses in Plants

Inhibitors of plant hormone transport