The exocyst, a eukaryotic tethering complex, coregulates targeted exocytosis as an effector of small GTPases in polarized cell growth. In land plants, several exocyst subunits are encoded by double or triple paralogs, culminating in tens of EXO70 paralogs. Out of 23 Arabidopsis thaliana EXO70 isoforms, we analyzed seven isoforms expressed in pollen. Genetic and microscopic analyses of single mutants in EXO70A2, EXO70C1, EXO70C2, EXO70F1, EXO70H3, EXO70H5, and EXO70H6 genes revealed that only a loss-of-function EXO70C2 allele resulted in a significant male-specific transmission defect (segregation 40%:51%:9%) due to aberrant pollen tube growth. Mutant pollen tubes grown in vitro exhibited an enhanced growth rate and a decreased thickness of the tip cell wall, causing tip bursts. However, exo70C2 pollen tubes could frequently recover and restart their speedy elongation, resulting in a repetitive stop-and-go growth dynamics. A pollen-specific depletion of the closest paralog, EXO70C1, using artificial microRNA in the exo70C2 mutant background, resulted in a complete pollen-specific transmission defect, suggesting redundant functions of EXO70C1 and EXO70C2. Both EXO70C1 and EXO70C2, GFP tagged and expressed under the control of their native promoters, localized in the cytoplasm of pollen grains, pollen tubes, and also root trichoblast cells. The expression of EXO70C2-GFP complemented the aberrant growth of exo70C2 pollen tubes. The absent EXO70C2 interactions with core exocyst subunits in the yeast two-hybrid assay, cytoplasmic localization, and genetic effect suggest an unconventional EXO70 function possibly as a regulator of exocytosis outside the exocyst complex. In conclusion, EXO70C2 is a novel factor contributing to the regulation of optimal tip growth of Arabidopsis pollen tubes.

Department of Experimental Botany Faculty of Science Charles University 12844 Prague 2 Czech Republic

Institute of Experimental Botany Czech Academy of Sciences 16502 Prague 6 Czech Republic ; and

Komentář v

Plant Physiol. 2017 May;174(1):3-4. doi: 10.1104/pp.17.00473. PubMed

Zobrazit více v PubMed

Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R, et al. (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301: 653–657 PubMed

Bashline L, Lei L, Li S, Gu Y (2014) Cell wall, cytoskeleton, and cell expansion in higher plants. Mol Plant 7: 586–600 PubMed

Bloch D, Pleskot R, Pejchar P, Potocký M, Trpkošová P, Cwiklik L, Vukašinović N, Sternberg H, Yalovsky S, Žárský V (2016) Exocyst SEC3 and phosphoinositides define sites of exocytosis in pollen tube initiation and growth. Plant Physiol 172: 980–1002 PubMed PMC

Chebli Y, Kaneda M, Zerzour R, Geitmann A (2012) The cell wall of the Arabidopsis pollen tube: spatial distribution, recycling, and network formation of polysaccharides. Plant Physiol 160: 1940–1955 PubMed PMC

Chebli Y, Kroeger J, Geitmann A (2013) Transport logistics in pollen tubes. Mol Plant 6: 1037–1052 PubMed

Cheung AY, Wu HM (2008) Structural and signaling networks for the polar cell growth machinery in pollen tubes. Annu Rev Plant Biol 59: 547–572 PubMed

Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735–743 PubMed

Cole RA, Fowler JE (2006) Polarized growth: maintaining focus on the tip. Curr Opin Plant Biol 9: 579–588 PubMed

Cole RA, McInally SA, Fowler JE (2014) Developmentally distinct activities of the exocyst enable rapid cell elongation and determine meristem size during primary root growth in Arabidopsis. BMC Plant Biol 14: 386. PubMed PMC

Cole RA, Synek L, Žárský V, Fowler JE (2005) SEC8, a subunit of the putative Arabidopsis exocyst complex, facilitates pollen germination and competitive pollen tube growth. Plant Physiol 138: 2005–2018 PubMed PMC

Cvrčková F, Bezvoda R, Zárský V (2010) Computational identification of root hair-specific genes in Arabidopsis. Plant Signal Behav 5: 1407–1418 PubMed PMC

Cvrčková F, Eliáš M, Hála M, Obermeyer G, Žárský V (2001) Small GTPases and conserved signalling pathways in plant cell morphogenesis: from exocytosis to exocyst. In Geitmann A, Cresti M, eds, Cell Biology of Plant and Fungal Tip Growth. IOS Press, Amsterdam, pp 105–122

Cvrčková F, Grunt M, Bezvoda R, Hála M, Kulich I, Rawat A, Zárský V (2012) Evolution of the land plant exocyst complexes. Front Plant Sci 3: 159. PubMed PMC

Dellago H, Löscher M, Ajuh P, Ryder U, Kaisermayer C, Grillari-Voglauer R, Fortschegger K, Gross S, Gstraunthaler A, Borth N, et al. (2011) Exo70, a subunit of the exocyst complex, interacts with SNEV(hPrp19/hPso4) and is involved in pre-mRNA splicing. Biochem J 438: 81–91 PubMed PMC

Drdová EJ, Synek L, Pečenková T, Hála M, Kulich I, Fowler JE, Murphy AS, Zárský V (2013) The exocyst complex contributes to PIN auxin efflux carrier recycling and polar auxin transport in Arabidopsis. Plant J 73: 709–719 PubMed

Edwards K, Johnstone C, Thompson C (1991) A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res 19: 1349. PubMed PMC

Eliáš M, Drdová E, Ziak D, Bavlnka B, Hála M, Cvrčková F, Soukupová H, Žárský V (2003) The exocyst complex in plants. Cell Biol Int 27: 199–201 PubMed

Fendrych M, Synek L, Pecenková T, Drdová EJ, Sekereš J, de Rycke R, Nowack MK, Zársky V (2013) Visualization of the exocyst complex dynamics at the plasma membrane of Arabidopsis thaliana. Mol Biol Cell 24: 510–520 PubMed PMC

Fendrych M, Synek L, Pecenková T, Toupalová H, Cole R, Drdová E, Nebesárová J, Sedinová M, Hála M, Fowler JE, et al. (2010) The Arabidopsis exocyst complex is involved in cytokinesis and cell plate maturation. Plant Cell 22: 3053–3065 PubMed PMC

Fu Y. (2015) The cytoskeleton in the pollen tube. Curr Opin Plant Biol 28: 111–119 PubMed

Grobei MA, Qeli E, Brunner E, Rehrauer H, Zhang R, Roschitzki B, Basler K, Ahrens CH, Grossniklaus U (2009) Deterministic protein inference for shotgun proteomics data provides new insights into Arabidopsis pollen development and function. Genome Res 19: 1786–1800 PubMed PMC

Guo W, Grant A, Novick P (1999) Exo84p is an exocyst protein essential for secretion. J Biol Chem 274: 23558–23564 PubMed

Hála M, Cole R, Synek L, Drdová E, Pecenková T, Nordheim A, Lamkemeyer T, Madlung J, Hochholdinger F, Fowler JE, et al. (2008) An exocyst complex functions in plant cell growth in Arabidopsis and tobacco. Plant Cell 20: 1330–1345 PubMed PMC

Heider MR, Munson M (2012) Exorcising the exocyst complex. Traffic 13: 898–907 PubMed PMC

Hepler PK, Rounds CM, Winship LJ (2013) Control of cell wall extensibility during pollen tube growth. Mol Plant 6: 998–1017 PubMed PMC

Hepler PK, Winship LJ (2015) The pollen tube clear zone: clues to the mechanism of polarized growth. J Integr Plant Biol 57: 79–92 PubMed

Hong D, Jeon BW, Kim SY, Hwang JU, Lee Y (2016) The ROP2-RIC7 pathway negatively regulates light-induced stomatal opening by inhibiting exocyst subunit Exo70B1 in Arabidopsis. New Phytol 209: 624–635 PubMed

Hou WC, Chang WH, Jiang CM (1999) Qualitative distinction of carboxyl group distributions in pectins with ruthenium red. Bot Bull Acad Sin 40: 115–119

Ito T, Motohashi R, Kuromori T, Mizukado S, Sakurai T, Kanahara H, Seki M, Shinozaki K (2002) A new resource of locally transposed Dissociation elements for screening gene-knockout lines in silico on the Arabidopsis genome. Plant Physiol 129: 1695–1699 PubMed PMC

Johnson-Brousseau SA, McCormick S (2004) A compendium of methods useful for characterizing Arabidopsis pollen mutants and gametophytically-expressed genes. Plant J 39: 761–775 PubMed

Kitashiba H, Liu P, Nishio T, Nasrallah JB, Nasrallah ME (2011) Functional test of Brassica self-incompatibility modifiers in Arabidopsis thaliana. Proc Natl Acad Sci USA 108: 18173–18178 PubMed PMC

Kleinboelting N, Huep G, Kloetgen A, Viehoever P, Weisshaar B (2012) GABI-Kat SimpleSearch: new features of the Arabidopsis thaliana T-DNA mutant database. Nucleic Acids Res 40: D1211–D1215 PubMed PMC

Kulich I, Cole R, Drdová E, Cvrcková F, Soukup A, Fowler J, Zárský V (2010) Arabidopsis exocyst subunits SEC8 and EXO70A1 and exocyst interactor ROH1 are involved in the localized deposition of seed coat pectin. New Phytol 188: 615–625 PubMed

Kulich I, Pečenková T, Sekereš J, Smetana O, Fendrych M, Foissner I, Höftberger M, Zárský V (2013) Arabidopsis exocyst subcomplex containing subunit EXO70B1 is involved in autophagy-related transport to the vacuole. Traffic 14: 1155–1165 PubMed

Kulich I, Vojtíková Z, Glanc M, Ortmannová J, Rasmann S, Žárský V (2015) Cell wall maturation of Arabidopsis trichomes is dependent on exocyst subunit EXO70H4 and involves callose deposition. Plant Physiol 168: 120–131 PubMed PMC

Kuromori T, Hirayama T, Kiyosue Y, Takabe H, Mizukado S, Sakurai T, Akiyama K, Kamiya A, Ito T, Shinozaki K (2004) A collection of 11 800 single-copy Ds transposon insertion lines in Arabidopsis. Plant J 37: 897–905 PubMed

Lai KS. (2016) Analysis of EXO70C2 expression revealed its specific association with late stages of pollen development. Plant Cell Tissue Organ Cult 124: 209–215

Lassig R, Gutermuth T, Bey TD, Konrad KR, Romeis T (2014) Pollen tube NAD(P)H oxidases act as a speed control to dampen growth rate oscillations during polarized cell growth. Plant J 78: 94–106 PubMed

Lavy M, Bloch D, Hazak O, Gutman I, Poraty L, Sorek N, Sternberg H, Yalovsky S (2007) A novel ROP/RAC effector links cell polarity, root-meristem maintenance, and vesicle trafficking. Curr Biol 17: 947–952 PubMed

Li S, Chen M, Yu D, Ren S, Sun S, Liu L, Ketelaar T, Emons AMC, Liu CM (2013) EXO70A1-mediated vesicle trafficking is critical for tracheary element development in Arabidopsis. Plant Cell 25: 1774–1786 PubMed PMC

Li S, van Os GM, Ren S, Yu D, Ketelaar T, Emons AMC, Liu CM (2010) Expression and functional analyses of EXO70 genes in Arabidopsis implicate their roles in regulating cell type-specific exocytosis. Plant Physiol 154: 1819–1830 PubMed PMC

Liu J, Guo W (2012) The exocyst complex in exocytosis and cell migration. Protoplasma 249: 587–597 PubMed

Loraine AE, McCormick S, Estrada A, Patel K, Qin P (2013) RNA-seq of Arabidopsis pollen uncovers novel transcription and alternative splicing. Plant Physiol 162: 1092–1109 PubMed PMC

MacAlister CA, Ortiz-Ramírez C, Becker JD, Feijó JA, Lippman ZB (2016) Hydroxyproline O-arabinosyltransferase mutants oppositely alter tip growth in Arabidopsis thaliana and Physcomitrella patens. Plant J 85: 193–208 PubMed PMC

Mayank P, Grossman J, Wuest S, Boisson-Dernier A, Roschitzki B, Nanni P, Nühse T, Grossniklaus U (2012) Characterization of the phosphoproteome of mature Arabidopsis pollen. Plant J 72: 89–101 PubMed

McKenna ST, Kunkel JG, Bosch M, Rounds CM, Vidali L, Winship LJ, Hepler PK (2009) Exocytosis precedes and predicts the increase in growth in oscillating pollen tubes. Plant Cell 21: 3026–3040 PubMed PMC

Mori T, Kuroiwa H, Higashiyama T, Kuroiwa T (2006) GENERATIVE CELL SPECIFIC 1 is essential for angiosperm fertilization. Nat Cell Biol 8: 64–71 PubMed

Moscatelli A, Idilli AI (2009) Pollen tube growth: a delicate equilibrium between secretory and endocytic pathways. J Integr Plant Biol 51: 727–739 PubMed

Nakagawa T, Kurose T, Hino T, Tanaka K, Kawamukai M, Niwa Y, Toyooka K, Matsuoka K, Jinbo T, Kimura T (2007) Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. J Biosci Bioeng 104: 34–41 PubMed

Pečenková T, Hála M, Kulich I, Kocourková D, Drdová E, Fendrych M, Toupalová H, Zársky V (2011) The role for the exocyst complex subunits Exo70B2 and Exo70H1 in the plant-pathogen interaction. J Exp Bot 62: 2107–2116 PubMed PMC

Pfeffer SR. (2013) Rab GTPase regulation of membrane identity. Curr Opin Cell Biol 25: 414–419 PubMed PMC

Picton JM, Steer MW (1983) Membrane recycling and the control of secretory activity in pollen tubes. J Cell Sci 63: 303–310 PubMed

Preuss D, Rhee SY, Davis RW (1994) Tetrad analysis possible in Arabidopsis with mutation of the QUARTET (QRT) genes. Science 264: 1458–1460 PubMed

Rounds CM, Lubeck E, Hepler PK, Winship LJ (2011) Propidium iodide competes with Ca2+ to label pectin in pollen tubes and Arabidopsis root hairs. Plant Physiol 157: 175–187 PubMed PMC

Rutley N, Twell D (2015) A decade of pollen transcriptomics. Plant Reprod 28: 73–89 PubMed PMC

Rybak K, Steiner A, Synek L, Klaeger S, Kulich I, Facher E, Wanner G, Kuster B, Žárský V, Persson S, et al. (2014) Plant cytokinesis is orchestrated by the sequential action of the TRAPPII and exocyst tethering complexes. Dev Cell 29: 607–620 PubMed

Safavian D, Zayed Y, Indriolo E, Chapman L, Ahmed A, Goring DR (2015) RNA silencing of exocyst genes in the stigma impairs the acceptance of compatible pollen in Arabidopsis. Plant Physiol 169: 2526–2538 PubMed PMC

Samuel MA, Chong YT, Haasen KE, Aldea-Brydges MG, Stone SL, Goring DR (2009) Cellular pathways regulating responses to compatible and self-incompatible pollen in Brassica and Arabidopsis stigmas intersect at Exo70A1, a putative component of the exocyst complex. Plant Cell 21: 2655–2671 PubMed PMC

Sanati Nezhad A, Packirisamy M, Geitmann A (2014) Dynamic, high precision targeting of growth modulating agents is able to trigger pollen tube growth reorientation. Plant J 80: 185–195 PubMed

Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, et al. (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9: 676–682 PubMed PMC

Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D (2006) Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell 18: 1121–1133 PubMed PMC

Sekereš J, Pejchar P, Šantrůček J, Vukašinović N, Žárský V, Potocký M (2017) Analysis of exocyst subunit EXO70 family reveals distinct membrane domains in tobacco pollen tubes. Plant Physiol 173: 1659–1675 PubMed PMC

Stegmann M, Anderson RG, Ichimura K, Pečenková T, Reuter P, Žársky V, McDowell JM, Shirasu K, Trujillo M (2012) The ubiquitin ligase PUB22 targets a subunit of the exocyst complex required for PAMP-triggered responses in Arabidopsis. Plant Cell 24: 4703–4716 PubMed PMC

Synek L, Schlager N, Eliás M, Quentin M, Hauser MT, Zárský V (2006) 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 48: 54–72 PubMed PMC

TerBush DR, Maurice T, Roth D, Novick P (1996) The exocyst is a multiprotein complex required for exocytosis in Saccharomyces cerevisiae. EMBO J 15: 6483–6494 PubMed PMC

TerBush DR, Novick P (1995) Sec6, Sec8, and Sec15 are components of a multisubunit complex which localizes to small bud tips in Saccharomyces cerevisiae. J Cell Biol 130: 299–312 PubMed PMC

Tu B, Hu L, Chen W, Li T, Hu B, Zheng L, Lv Z, You S, Wang Y, Ma B, et al. (2015) Disruption of OsEXO70A1 causes irregular vascular bundles and perturbs mineral nutrient assimilation in rice. Sci Rep 5: 18609. PubMed PMC

Vega IE, Hsu SC (2001) The exocyst complex associates with microtubules to mediate vesicle targeting and neurite outgrowth. J Neurosci 21: 3839–3848 PubMed PMC

Vukašinović N, Cvrčková F, Eliáš M, Cole R, Fowler JE, Žárský V, Synek L (2014) Dissecting a hidden gene duplication: the Arabidopsis thaliana SEC10 locus. PLoS ONE 9: e94077. PubMed PMC

Vukašinović N, Oda Y, Pejchar P, Synek L, Pečenková T, Rawat A, Sekereš J, Potocký M, Žárský V (2016) Microtubule-dependent targeting of the exocyst complex is necessary for the xylem development in Arabidopsis. New Phytol 213: 1052–1067 PubMed

Vukašinović N, Žárský V (2016) Tethering complexes in the Arabidopsis endomembrane system. Front Cell Dev Biol 4: 46. PubMed PMC

Wang S, Liu Y, Adamson CL, Valdez G, Guo W, Hsu SC (2004) The mammalian exocyst, a complex required for exocytosis, inhibits tubulin polymerization. J Biol Chem 279: 35958–35966 PubMed

Wei LQ, Xu WY, Deng ZY, Su Z, Xue Y, Wang T (2010) Genome-scale analysis and comparison of gene expression profiles in developing and germinated pollen in Oryza sativa. BMC Genomics 11: 338. PubMed PMC

Winter D, Vinegar B, Nahal H, Ammar R, Wilson GV, Provart NJ (2007) An “Electronic Fluorescent Pictograph” browser for exploring and analyzing large-scale biological data sets. PLoS ONE 2: e718. PubMed PMC

Wu H, Rossi G, Brennwald P (2008) The ghost in the machine: small GTPases as spatial regulators of exocytosis. Trends Cell Biol 18: 397–404 PubMed PMC

Žárský V, Cvrcková F, Potocký M, Hála M (2009) Exocytosis and cell polarity in plants: exocyst and recycling domains. New Phytol 183: 255–272 PubMed

Žárský V, Kulich I, Fendrych M, Pečenková T (2013) Exocyst complexes multiple functions in plant cells secretory pathways. Curr Opin Plant Biol 16: 726–733 PubMed

Žárský V, Potocký M (2010) Recycling domains in plant cell morphogenesis: small GTPase effectors, plasma membrane signalling and the exocyst. Biochem Soc Trans 38: 723–728 PubMed

Zhang C, Brown MQ, van de Ven W, Zhang ZM, Wu B, Young MC, Synek L, Borchardt D, Harrison R, Pan S, et al. (2016) Endosidin2 targets conserved exocyst complex subunit EXO70 to inhibit exocytosis. Proc Natl Acad Sci USA 113: E41–E50 PubMed PMC

Zhang X, Pumplin N, Ivanov S, Harrison MJ (2015) EXO70I is required for development of a sub-domain of the periarbuscular membrane during arbuscular mycorrhizal symbiosis. Curr Biol 25: 2189–2195 PubMed

Zhao Y, Liu J, Yang C, Capraro BR, Baumgart T, Bradley RP, Ramakrishnan N, Xu X, Radhakrishnan R, Svitkina T, et al. (2013) Exo70 generates membrane curvature for morphogenesis and cell migration. Dev Cell 26: 266–278 PubMed PMC

Zimmermann P, Hirsch-Hoffmann M, Hennig L, Gruissem W (2004) GENEVESTIGATOR: Arabidopsis microarray database and analysis toolbox. Plant Physiol 136: 2621–2632 PubMed PMC

Zonia L, Munnik T (2008) Vesicle trafficking dynamics and visualization of zones of exocytosis and endocytosis in tobacco pollen tubes. J Exp Bot 59: 861–873 PubMed

Zonia L, Munnik T (2009) Uncovering hidden treasures in pollen tube growth mechanics. Trends Plant Sci 14: 318–327 PubMed

Zuo X, Zhang J, Zhang Y, Hsu SC, Zhou D, Guo W (2006) Exo70 interacts with the Arp2/3 complex and regulates cell migration. Nat Cell Biol 8: 1383–1388 PubMed

Plasma membrane phospholipid signature recruits the plant exocyst complex via the EXO70A1 subunit

LARP6C orchestrates posttranscriptional reprogramming of gene expression during hydration to promote pollen tube guidance

Functional Specialization within the EXO70 Gene Family in Arabidopsis

The Arabidopsis thaliana Class II Formin FH13 Modulates Pollen Tube Growth

Transcriptomic and Proteomic Insights into Amborella trichopoda Male Gametophyte Functions

EXO70A2 Is Critical for Exocyst Complex Function in Pollen Development

Synergy among Exocyst and SNARE Interactions Identifies a Functional Hierarchy in Secretion during Vegetative Growth

Redundant and Diversified Roles Among Selected Arabidopsis thaliana EXO70 Paralogs During Biotic Stress Responses

Regulation of Exocyst Function in Pollen Tube Growth by Phosphorylation of Exocyst Subunit EXO70C2