, Volume 252, Issue 1, pp 63–75 | Cite as

In vitro inhibition of incompatible pollen tubes in Nicotiana alata involves the uncoupling of the F-actin cytoskeleton and the endomembrane trafficking system

  • Juan A. Roldán
  • Hernán J. Rojas
  • Ariel GoldraijEmail author
Original Article


In the S-RNase-based self-incompatibility system, subcellular events occurring in the apical region of incompatible pollen tubes during the pollen rejection process are poorly understood. F-actin dynamics and endomembrane trafficking are crucial for polar growth, which is temporally and spatially controlled in the tip region of pollen tubes. Thus, we developed a simple in vitro assay to study the changes in the F-actin cytoskeleton and the endomembrane system at the apical region of incompatible pollen tubes in Nicotiana alata. Growth but not germination of pollen tubes of S c10 -, S 70 -, and S 75 -haplotypes was selectively inhibited by style extracts carrying the same haplotypes. Pollen F-actin cytoskeleton and endomembrane system, visualized by fluorescent markers, were normal during the initial 60 min of pollen culture in the presence of compatible and incompatible style extracts. Additional culture resulted in complete growth arrest and critical alterations in the integrity of the F-actin cytoskeleton and the endomembrane system of incompatible pollen tubes. The F-actin ring and the V-shaped zone disappeared from the apical region, while distorted F-actin cables and progressive formation of membrane aggregates evolved in the subapical region and the shank. The vacuolar network of incompatible pollen tubes invaded the tip region, but vacuolar membrane integrity remained mostly unaffected. The polar growth machinery of incompatible pollen tubes was uncoupled, as evidenced by the severe disruption of colocalization between the F-actin cytoskeleton and the endomembrane compartments. A model of pollen rejection integrating the main subcellular events occurring in incompatible pollen is discussed.


Endomembrane system F-actin cytoskeleton Nicotiana alata Polar growth Self-incompatibility 



Actin binding proteins


Growth medium




Vacuolar pyrophosphatase



We thank Dr Carlos Mas and Dr Cecilia Sampedro for their assistance in digital image processing and Gabriela Diaz Cortez for editorial and language assistance. The vPPase antibody was kindly provided by Dr Thomas Phillips (University of Missouri). This work was supported by funds from the following Argentina state agencies: Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT; PICT 32933); Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET; PIP 11220090100265); and Secretaría de Ciencia y Técnica-Universidad Nacional de Córdoba (SECyT-UNC; 05/C466) to AG. AG is a member of CONICET. JAR has a CONICET postdoctoral scholarship. HJR is a SECyT-Universidad Nacional de Córdoba scholarship holder.

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

709_2014_658_Fig9_ESM.gif (32 kb)
Fig. S1

Pollen of S c10 -, S 70 -, and, S 75 - haplotypes were cultured in GM. Percentages of germinated (white) and non-germinated (grey) pollen grains are shown. The values are the average ± SD of three or more independent experiments. (GIF 32 kb)

709_2014_658_MOESM1_ESM.eps (790 kb)
High Resolution Image (EPS 790 kb)
709_2014_658_Fig10_ESM.gif (86 kb)
Fig. S2

In vitro growth of Nicotiana alata pollen tubes challenged with compatible and incompatible style extract. Pollen tubes were cultured for 6 h in the presence of compatible (white) and incompatible (gray) style extracts (a-c). The pollen S c10 - and the style extract S c10 S c10 in a were prepared from different S c10 S c10 individuals. The values are the average ± SD of three or more independent experiments. Data were statistically analyzed using one-way ANOVA followed by Tukey test. ***P<0,001. (GIF 85 kb)

709_2014_658_MOESM2_ESM.eps (1.9 mb)
High Resolution Image (EPS 1899 kb)
709_2014_658_Fig11_ESM.gif (85 kb)
Fig. S3

Patterns of F-actin cytoskeleton disorganization after 180 m of culture with incompatible style extract. Images are confocal projections of F-actin stained with fluorescent phalloidin. a F-actin cables distorted and slightly fragmented. b F-actin cables severely fragmented. c F-actin foci. Patterns a, b, and c represented 88%, 9%, and 3%, respectively, of the total pollen tubes analyzed. Bars 10 μm. (GIF 85 kb)

709_2014_658_MOESM3_ESM.tif (2 mb)
High Resolution Image (TIFF 2071 kb)
video online resource 1

Elongation rate and vesicle dynamics of a pollen tube started to be tracked 50 min after challenging with a compatible style extract. (MPG 8824 kb)

video online resource 2

Elongation rate and vesicle dynamics of a pollen tube started to be tracked 50 min after challenging with an incompatible style extract. (MPG 6998 kb)


  1. Beale KM, Johnson MA (2013) Speed dating, rejection, and finding the perfect mate: advice from flowering plants. Curr Opin Plant Biol 16:590–597PubMedCrossRefGoogle Scholar
  2. Bosch M, Franklin-Tong VE (2007) Temporal and spatial activation of a caspase-like enzymes induced by self-incompatibility in Papaver pollen. Proc Natl Acad Sci U S A 104:18327–18332PubMedCentralPubMedCrossRefGoogle Scholar
  3. Chebli Y, Kroeger J, Geitmann A (2013) Transport logistic in pollen tubes. Mol Plant 6:1037–1052PubMedCrossRefGoogle Scholar
  4. Chen T, Teng N, Wu X, Wang Y, Tang W, Šamaj J, Baluška F, Lin J (2007) Disruption of actin filaments by latrunculin B affects cell wall construction in Picea meyeri pollen tube by disturbing vesicle trafficking. Plant Cell Physiol 48:19–30PubMedCrossRefGoogle Scholar
  5. Cheung AY, Wu HM (2008) Structural and signaling networks for the polar cell growth machinery in pollen tubes. Annu Rev Plant Biol 59:547–572PubMedCrossRefGoogle Scholar
  6. Cheung AY, Niroomand S, Zou Y, Wu HM (2010) A transmembrane formin nucleates subapical actin assembly and controls tip-focused growth in pollen tubes. Proc Natl Acad Sci U S A 107:16390–16395PubMedCentralPubMedCrossRefGoogle Scholar
  7. de Graaf BHJ, Cheung AY, Andreyeva T, Lavasseur K, Kieliszewski M, Wu HM (2005) Rab11 GTPase-regulated membrane trafficking is crucial for tip-focused pollen tube growth in tobacco. Plant Cell 17:2564–2579PubMedCentralPubMedCrossRefGoogle Scholar
  8. Franklin-Tong VE (2008) Self-incompatibility in Papaver rhoeas: progress in understanding mechanisms involved in regulating self-incompatibility in Papaver. In: Franklin-Tong VE (ed) Self-incompatibility in flowering plants—evolution, diversity and mechanisms. Springer-Verlag, Berlin, pp 237–258CrossRefGoogle Scholar
  9. Fu Y, Wu G, Yang Z (2001) Rop GTPase-dependent dynamics of tip localized F-actin controls tip growth in pollen tubes. J Cell Biol 152:1019–1032PubMedCentralPubMedCrossRefGoogle Scholar
  10. Geitmann A, Snowman BN, Emons AMC, Franklin-Tong VE (2000) Alterations in the actin cytoskeleton of pollen tubes are induced by the self-incompatibility reaction in Papaver rhoeas. Plant Cell 12:1239–1251PubMedCentralPubMedCrossRefGoogle Scholar
  11. Gibbon BC, Kovar DR, Staiger CJ (1999) Latrunculin B has different effects on pollen germination and tube growth. Plant Cell 11:2349–2364PubMedCentralPubMedCrossRefGoogle Scholar
  12. Goldraij A, Kondo K, Lee CB, Hancock CN, Sivaguru M, Vazquez-Santana S, Kim S, Phillips TE, Cruz-Garcia F, McClure BA (2006) Compartmentalization of S-RNase and HT-B degradation in self-incompatible Nicotiana. Nature 439:805–810PubMedCrossRefGoogle Scholar
  13. Goldraij A, Roldán JA, Rojas HJ (2012) Early F-actin disorganization may be signaling vacuole disruption in incompatible pollen tubes of Nicotiana alata. Plant Signal Behav 7:1695–1697PubMedCentralPubMedCrossRefGoogle Scholar
  14. Gray JE, McClure BA, Bönig I, Anderson MA, Clarke AE (1991) Action of the style product of the self-incompatibility gene of Nicotiana alata (S-RNase) on in vitro-grown pollen tubes. Plant Cell 3:271–283PubMedCentralPubMedCrossRefGoogle Scholar
  15. Guan Y, Guo J, Li H, Yang Z (2013) Signaling in pollen tube growth: crosstalk, feedback, and missing links. Mol Plant 6:1053–1064PubMedCentralPubMedCrossRefGoogle Scholar
  16. Hancock CN, Kent L, McClure BA (2005) The stylar 120 kDa glycoprotein is required for S-specific pollen rejection in Nicotiana. Plant J 43:716–723PubMedCrossRefGoogle Scholar
  17. Hiratsuka S, Zhang SL, Nakagawa E, Kawai Y (2001) Selective inhibition of the growth of incompatible pollen tubes by S-protein in the Japanese pear. Sex Plant Reprod 13:209–215CrossRefGoogle Scholar
  18. Hörmanseder K, Obermeyer G, Foissner I (2005) Disturbance of endomembrane trafficking by brefeldin A and calyculin A reorganizes the actin cytoskeleton of Lilium longiflorum pollen tubes. Protoplasma 227:25–36PubMedCrossRefGoogle Scholar
  19. Hua Z, Fields A, T-h K (2008) Biochemical models for S-RNase-based self-incompatibility. Mol Plant 1:575–585PubMedCrossRefGoogle Scholar
  20. Huang S, Blanchoin L, Chaudhry F, Franklin-Tong VE, Staiger CJ (2004) A gelsolin-like protein from Papaver rhoeas pollen (PrABP80) stimulates calcium-regulated severing and depolymerization of actin filaments. J Biol Chem 279:23364–23375PubMedCrossRefGoogle Scholar
  21. Iwano M, Takayama S (2011) Self/non-self discrimination in angiosperm self-incompatibility. Curr Opin Plant Biol 15:1–6CrossRefGoogle Scholar
  22. Jiménez-Durán K, McClure B, García-Campusano F, Rodríguez-Sotres R, Cisneros J, Busot G, Cruz-García F (2013) NaStEP: a proteinase inhibitor essential to self-incompatibility and a positive regulator of HT-B stability in Nicotiana alata pollen tubes. Plant Physiol 161:97–107PubMedCentralPubMedCrossRefGoogle Scholar
  23. Kost B, Lemichez E, Spielhofer P, Hong Y, Tolias K, Carpenter C, Chua NH (1999) Rac homologues and compartmentalized phosphatidylinositol 4, 5-bisphosphate act in a common pathway to regulate polar pollen tube growth. J Cell Biol 145:317–330PubMedCentralPubMedCrossRefGoogle Scholar
  24. Kumar A, McClure BA (2010) Pollen–pistil interactions and the endomembrane system. J Exp Bot 61:2001–2013PubMedCrossRefGoogle Scholar
  25. Lee YJ, Szumlanski A, Nielsen E, Yang Z (2008) Rho-GTPase-dependent filamentous actin dynamics coordinate vesicle targeting and exocytosis during tip growth. J Cell Biol 181:1155–1168PubMedCentralPubMedCrossRefGoogle Scholar
  26. Liu Z, Xu G, Zhang S (2007) Pyrus pyrifolia stylar S-RNase induces alterations in the actin cytoskeleton in self-pollen and tubes in vitro. Protoplasma 232:61–67PubMedCrossRefGoogle Scholar
  27. Luu DT, Qin X, Morse D, Cappadocia M (2000) S-RNase uptake by compatible pollen tubes in gametophytic self-incompatibility. Nature 407:649–651PubMedCrossRefGoogle Scholar
  28. McClure BA, Mou B, Canevascini S, Bernatzky R (1999) A small asparagine-rich protein required for S-allele-specific pollen rejection in Nicotiana. Proc Natl Acad Sci U S A 96:13548–13553PubMedCentralPubMedCrossRefGoogle Scholar
  29. McClure BA (2008) Self-incompatibility in Papaver rhoeas: progress in understanding mechanisms involved in regulating self-incompatibility in Papaver. In: Franklin-Tong VE (ed) Self-incompatibility in flowering plants—evolution, diversity and mechanisms. Springer-Verlag, Berlin, pp 217–236CrossRefGoogle Scholar
  30. Moscatelli A, Ciampolini F, Rodighiero S, Onelli E, Cresti M, Santo N, Idilli A (2007) Distinct endocytic pathways identified in tobacco pollen tubes using charged nanogold. J Cell Sci 120:3804–3819PubMedCrossRefGoogle Scholar
  31. Poulter NS, Staiger CJ, Rappoport JZ, Franklin-Tong VE (2010) Actin-binding proteins implicated in the formation of the punctate actin foci stimulated by the self-incompatibility response in Papaver. Plant Physiol 152:1274–1283PubMedCentralPubMedCrossRefGoogle Scholar
  32. Onelli E, Moscatelli A (2013) Endocytic pathways and recycling in growing pollen tubes. Plants 2:211–229CrossRefGoogle Scholar
  33. Qiao H, Huang H, Zhao L, Zhou J, Huang J, Zhang Y, Xue Y (2004) The F-Box protein AhSLF-S2 physically interacts with S-RNases that may be inhibited by the ubiquitin/26S proteasome pathway of protein degradation during compatible pollination in Antirrhinum. Plant Cell 16:582–595PubMedCentralPubMedCrossRefGoogle Scholar
  34. Qin Y, Yang Z (2011) Rapid tip growth: insights from pollen tubes. Semin Cell Dev Biol 22:816–824PubMedCentralPubMedCrossRefGoogle Scholar
  35. Qu X, Zhang H, Xie Y, Wang J, Chen N, Huang S (2013) Arabidopsis villins promote actin turnover at pollen tube tips and facilitate the construction of actin collars. Plant Cell 25:1803–1817PubMedCentralPubMedCrossRefGoogle Scholar
  36. Rea AC, Nasrallah JB (2008) Self-incompatibility systems: barriers to self-fertilization in flowering plants. Int J Dev Biol 52:627–636PubMedCrossRefGoogle Scholar
  37. Roldán JA, Quiroga R, Goldraij A (2010) Molecular and genetic characterization of novel S-RNases from a natural population of Nicotiana alata. Plant Cell Rep 29:735–746Google Scholar
  38. Roldán JA, Rojas HJ, Goldraij A (2012) Disorganization of F-actin cytoskeleton precedes vacuolar disruption in pollen tubes during the in vivo self-incompatibility response in Nicotiana alata. Ann Bot 110:787–795PubMedCentralPubMedCrossRefGoogle Scholar
  39. Serrano I, Olmedilla A (2012) Histochemical location of key enzyme activities involved in receptivity and self-incompatibility in the olive tree (Olea europaea L.). Plant Sci 197:40–49PubMedCrossRefGoogle Scholar
  40. Snowman BN, Kovar DR, Shevchenko G, Franklin-Tong VE, Staiger CJ (2002) Signal-mediated depolymerization of actin in pollen during the self-incompatibility response. Plant Cell 14:2613–2626PubMedCentralPubMedCrossRefGoogle Scholar
  41. Staiger CJ, Poulter NS, Henty JL, Franklin-Tong VE, Blanchoin L (2010) Regulation of actin dynamics by actin-binding proteins in pollen. J Exp Bot 61:1969–1986PubMedCrossRefGoogle Scholar
  42. Su H, Zhu J, Cai C, Pei W, Huang J, Dong H, Ren H (2012) FIMBRIN1 is involved in lily pollen tube growth by stabilizing the actin fringe. Plant Cell 24:4539–4554PubMedCentralPubMedCrossRefGoogle Scholar
  43. Thomas SG, Franklin-Tong VE (2004) Self-incompatibility triggers programmed cell death in Papaver pollen. Nature 429:305–309PubMedCrossRefGoogle Scholar
  44. Thomas SG, Huang S, Li S, Staiger CJ, Franklin-Tong VE (2006) Actin depolymerization is sufficient to induce programmed cell death in self-incompatible pollen. J Cell Biol 174:221–229PubMedCentralPubMedCrossRefGoogle Scholar
  45. Vidali L, McKenna ST, Hepler PK (2001) Actin polymerization is essential for pollen tube growth. Mol Biol Cell 12:2534–2545PubMedCentralPubMedCrossRefGoogle Scholar
  46. Wang HY, Xue Y (2005) Subcellular localization of the S-locus F-box protein AhSLF-S2 in pollen and pollen tubes of self-incompatible Antirrhinum. J Integr Plant Biol 47:76–83CrossRefGoogle Scholar
  47. Wang CL, Zhang SL (2011) A cascade signal pathway occurs in self-incompatibility of Pyrus pyrifolia. Plant Sign Behav 6:420–421Google Scholar
  48. Wang CL, Xu GH, Jiang XT, Chen G, Wu J, Wu HQ, Zhang SL (2009) S-RNase triggers mitochondrial alteration and DNA degradation in the incompatible pollen tube of Pyrus pyrifolia in vitro. Plant J 57:220–229PubMedCrossRefGoogle Scholar
  49. Wang CL, Wu J, Xu GH, Gao YB, Chen G, Wu JI, Wu HQ, Zhang SL (2010) S-RNase disrupts tip-localized reactive oxygen species and induces nuclear DNA degradation in incompatible pollen tubes of Pyrus pyrifolia. J Cell Sci 123:4301–4309PubMedCrossRefGoogle Scholar
  50. Wang H, Zhuang X, Cai Y, Cheung AY, Jiang L (2013) Apical F-actin-regulated exocytic targeting of NtPPME1 is essential for construction and rigidity of the pollen tube cell wall. Plant J 76:367–379PubMedCrossRefGoogle Scholar
  51. Wheeler MJ, de Graaf BH, Hadjiosif N, Perry RM, Poulter NS, Osman K, Vatovec S, Harper A, Franklin FC, Franklin-Tong VE (2009) Identification of the pollen self-incompatibility determinant in Papaver rhoeas. Nature 459:992–995PubMedCentralPubMedCrossRefGoogle Scholar
  52. Wilkins KA, Poulter NS, Franklin-Tong VE (2014) Taking one for the team: self-recognition and cell suicide in pollen. J Exp Bot. doi: 10.1093/jxb/ert468 Google Scholar
  53. Zhang Y, He J, Lee D, McCormick S (2010) Interdependence of endomembrane trafficking and actin dynamics during polarized growth of Arabidopsis pollen tubes. Plant Physiol 152:2200–2210PubMedCentralPubMedCrossRefGoogle Scholar
  54. Zonia L, Munnik T (2008) Vesicle trafficking dynamics and visualization of zones of exocytosis and endocytosis in tobacco pollen tubes. J Exp Bot 59:861–873PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2014

Authors and Affiliations

  • Juan A. Roldán
    • 1
  • Hernán J. Rojas
    • 1
  • Ariel Goldraij
    • 1
    Email author
  1. 1.Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC–CONICET), Departamento de Química Biológica, Facultad de Ciencias QuímicasUniversidad Nacional de CórdobaCórdobaArgentina

Personalised recommendations