, Volume 242, Issue 1–4, pp 19–33 | Cite as

Dynamic morphologies of pollen plastids visualised by vegetative-specific FtsZ1–GFP in Arabidopsis thaliana

  • Makoto T. FujiwaraEmail author
  • Haruki Hashimoto
  • Yusuke Kazama
  • Tomonari Hirano
  • Yasushi Yoshioka
  • Seishiro Aoki
  • Naoki Sato
  • Ryuuichi D. Itoh
  • Tomoko Abe
Original Article


The behaviour and multiplication of pollen plastids have remained elusive despite their crucial involvement in cytoplasmic inheritance. Here, we present live images of plastids in pollen grains and growing tubes from transgenic Arabidopsis thaliana lines expressing stroma-localised FtsZ1–green-fluorescent protein fusion in a vegetative cell-specific manner. Vegetative cells in mature pollen contained a morphologically heterogeneous population of round to ellipsoidal plastids, whilst those in late-developing (maturing) pollen included plastids that could have one or two constriction sites. Furthermore, plastids in pollen tubes exhibited remarkable tubulation, stromule (stroma-filled tubule) extension, and back-and-forth movement along the direction of tube growth. Plastid division, which involves the FtsZ1 ring, was rarely observed in mature pollen grains.


Arabidopsis thaliana FtsZ ring Leucoplast Plastid division Stromule 



The authors are grateful to Prof. Danny J. Schnell (University of Massachusetts at Amherst, USA) for the gift of antiserum against Arabidopsis Tic110, Dr. Yasuo Niwa (University of Shizuoka, Japan) for the original GFP vector, Arabidopsis Biological Resource Center (Ohio State University, USA) for providing the arc11 seeds and Prof. Rachel M. Leech and Ms. Joanne L. Marrison (University of York, UK) for donating them. We also thank Dr. Yukihisa Shimada (RIKEN Plant Science Center, Japan), Dr. Takeshi Nakano, Ms. Sumie Ohbu (RIKEN), Mr. Tadasuke Chiba (KS Olympus, Japan) and the facility of RIKEN Radioactive Isotope Beam Factory for supporting this study. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 17780077 to M.T.F., No. 20657015 to R.D.I.) and the Agricultural Chemical Research Foundation (to M.T.F. and R.D.I.).

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

709_2010_119_MOESM1_ESM.mpg (194 kb)
Movie S1 Time-lapse observation of FtsZ1–GFP in a mature wild-type pollen grain. Fluorescence images were taken for 60 s at 4-s intervals; 60 s corresponds to 1.5 s in this movie. Note that pollen was incubated on in vitro germination medium before observation. (MPG 194 kb)
709_2010_119_MOESM2_ESM.mpg (210 kb)
Movie S2 Time-lapse observation of FtsZ1–GFP in a mature wild-type pollen grain. Fluorescence images were taken for 100 s at 4-s intervals; 100 s corresponds to 2.5 s in this movie. Note that the pollen sample is same with that of Fig. 2h and was not incubated before observation. (MPG 210 kb)
709_2010_119_MOESM3_ESM.mpg (396 kb)
Movie S3 Time-lapse observation of a mature wild-type pollen grain. DIC images were taken for 76 s at 4-s intervals; 76 s corresponds to 1.9 s in this movie. Note that pollen was incubated on in vitro germination medium before observation. (MPG 396 kb)
709_2010_119_MOESM4_ESM.mpg (142 kb)
Movie S4 Z-series images of FtsZ1–GFP in a maturing pollen grain of the wild type. Fluorescence images taken for 60 s at 4-s intervals are shown in this movie. (MPG 142 kb)
709_2010_119_MOESM5_ESM.mpg (286 kb)
Movie S5 Time-lapse observation of FtsZ1–GFP in maturing wild-type pollen grain. Fluorescence images were taken for 120 s at 4-s intervals; 120 s corresponds to 3.0 s in this movie. The pollen sample is the same as that in Fig. 3c. (MPG 286 kb)
709_2010_119_MOESM6_ESM.mpg (164 kb)
Movie S6 Time-lapse observation of FtsZ1–GFP in a germinating wild-type pollen grain. Fluorescence images were taken for 80 s at 4-s intervals; 80 s corresponds to 2.0 s in this movie. Germination is just about to occur at the left part of the grain. The pollen sample is the same as that in Fig. 4b. (MPG 164 kb)
Movie S7

Time-lapse observation of FtsZ1–GFP in a germinating pollen grain of the wild-type. Fluorescence images were taken for 120 s at 4-s intervals. Germination has occurred at the left part of grain; 120 s corresponds to 3.0 s in this movie. The pollen sample is the same as that in Fig. 4d. (MPG 380 kb)

709_2010_119_MOESM8_ESM.mpg (268 kb)
Movie S8 Time-lapse observation of a germinating wild-type pollen tube. DIC images were taken for 132 s at 4-s intervals; 132 s corresponds to 3.3 s in this movie. (MPG 268 kb)
Movie S9

Time-lapse observation of FtsZ1–GFP in germinated wild-type pollen. Fluorescence images were taken for 116 s at 4-s intervals. 116 s correspond to 2.9 s in this movie. The pollen sample is the same as that in Fig. 4f. (MPG 288 kb)

709_2010_119_MOESM10_ESM.mpg (348 kb)
Movie S10 Time-lapse observation of the growing tip of the wild-type pollen tube. DIC images were taken for 128 s at 4-s intervals; 128 s correspond to 3.2 s in this movie. (MPG 348 kb)
709_2010_119_MOESM11_ESM.mpg (1.3 mb)
Movie S11 Time-lapse observation of FtsZ1–GFP in the pollen tube of the wild type. Fluorescence images were taken for 136 s at 4-s intervals. Tip growth (the arrow shows the tip point) was in the right-to-left direction; 136 s correspond to 3.4 s in this movie. The pollen sample is the same as that in Fig. 4j. (MPG 1326 kb)
709_2010_119_MOESM12_ESM.mpg (358 kb)
Movie S12 Time-lapse observation of FtsZ1–GFP in a wild-type pollen tube. Fluorescence images were taken for 195 s at 5-s intervals. Tip growth was in the left-to-right direction; 195 s corresponds to 3.9 s in this movie. (MPG 358 kb)
Movie S13

Z-series images of FtsZ1–GFP in a mature pollen grain of arc11. Fluorescence images taken for 52 s at 4-s intervals are shown in this movie. The pollen sample is the same as that in Fig. 5b. (MPG 232 kb)

709_2010_119_MOESM14_ESM.mpg (124 kb)
Movie S14 Time-lapse observation of FtsZ1–GFP in a mature pollen grain of arc11. Fluorescence images were taken for 51 s at 3-s intervals; 51 s corresponds to 1.7 s in this movie. (MPG 124 kb)
709_2010_119_MOESM15_ESM.mpg (402 kb)
Movie S15 Time-lapse observation of FtsZ1–GFP in a pollen tube of arc11. Fluorescence images were taken for 72 s at 4-s intervals. A whole picture of germinated pollen is presented; 72 s corresponds to 1.5 s in this movie. (MPG 402 kb)


  1. Boavida LC, McCormick S (2007) Temperature as a determinant factor for increased and reproducible in vitro pollen germination in Arabidopsis thaliana. Plant J 52:570–582CrossRefPubMedGoogle Scholar
  2. Cai G, Cresti M (2009) Organelle motility in the pollen tube: a tale of 20 years. J Exp Bot 60:495–508CrossRefPubMedGoogle Scholar
  3. Chen Y, Asano T, Fujiwara MT, Yoshida S, Machida Y, Yoshioka Y (2009) Plant cells without detectable plastids are generated in the crumpled leaf mutant of Arabidopsis thaliana. Plant Cell Physiol 50:956–969CrossRefPubMedGoogle Scholar
  4. Cheung AY, Chen CY, Glaven RH, de Graaf BHJ, Vidali L, Hepler PK, Wu HM (2002) Rab2 GTPase regulates vesicle trafficking between the endoplasmic reticulum and the Golgi bodies and is important to pollen tube growth. Plant Cell 14:945–962CrossRefPubMedGoogle Scholar
  5. Clément C, Chavant L, Burrus M, Audran JC (1994) Anther starch variations in Lilium during pollen development. Sex Plant Reprod 7:347–356CrossRefGoogle Scholar
  6. Colletti KS, Tattersall EA, Pyke KA, Froelich JE, Stokes KD, Osteryoung KW (2000) A homologue of the bacterial cell division site-determining factor MinD mediates placement of the chloroplast division apparatus. Curr Biol 10:507–516CrossRefPubMedGoogle Scholar
  7. de Graaf BHJ, Cheung AY, Andreyeva T, Levasseur K, Kieliszewski M, Wu HM (2005) Rab11 GTPase-regulated membrane trafficking is crucial for tip-focused pollen tube growth in tobacco. Plant Cell 17:2564–2579CrossRefPubMedGoogle Scholar
  8. Forth D, Pyke KA (2006) The suffulta mutation in tomato reveals a novel method of plastid replication during fruit ripening. J Exp Bot 57:1971–1979CrossRefPubMedGoogle Scholar
  9. Friml J, Benková E, Mayer U, Palme K, Muster G (2003) Automated whole mount localisation techniques for plant seedlings. Plant J 34:115–124CrossRefPubMedGoogle Scholar
  10. Fujiwara MT, Nakamura A, Itoh R, Shimada Y, Yoshida S, Møller SG (2004) Chloroplast division site placement requires dimerisation of the ARC11/AtMinD1 protein in Arabidopsis. J Cell Sci 117:2399–2410CrossRefPubMedGoogle Scholar
  11. Fujiwara MT, Hashimoto H, Kazama Y, Abe T, Yoshida S, Sato N, Itoh RD (2008) The assembly of the FtsZ ring at the mid-chloroplast division site depends on a balance between the activities of AtMinE1 and ARC11/AtMinD1. Plant Cell Physiol 49:345–361CrossRefPubMedGoogle Scholar
  12. Fujiwara MT, Li D, Kazama Y, Abe T, Uno T, Yamagata Y, Kanamaru K, Itoh RD (2009a) Further evaluation of the localisation and functionality of hemagglutinin epitope- and fluorescent protein-tagged AtMinD1 in Arabidopsis thaliana. Biosci Biotechnol Biochem 73:1693–1697CrossRefPubMedGoogle Scholar
  13. Fujiwara MT, Sekine K, Yamamoto YY, Abe T, Sato N, Itoh RD (2009b) Live imaging of chloroplast FtsZ1 filaments, rings, spirals, and motile dot structures in the AtMinE1 mutant and overexpressor of Arabidopsis thaliana. Plant Cell Physiol 50:1116–1126CrossRefPubMedGoogle Scholar
  14. Gao H, Kadirjan-Kalbach D, Froehlich JE, Osteryoung KW (2003) ARC5, a cytosolic dynamin-like protein from plants, is part of the chloroplast division machinery. Proc Natl Acad Sci USA 100:4328–4333CrossRefPubMedGoogle Scholar
  15. Gunning BES (2005) Plastid stromules: video microscopy of their outgrowth, retraction, tensioning, anchoring, branching, bridging, and tip-shedding. Protoplasma 225:33–42CrossRefPubMedGoogle Scholar
  16. Hagemann R (2004) The sexual inheritance of plant organelles. In: Daniell H, Chase C (eds) Molecular Biology and Biotechnology of Plant Organelles. Springer, Dordrecht, The Netherlands, pp 93–113CrossRefGoogle Scholar
  17. Heslop-Harrison J, Heslop-Harrison Y (1990) Dynamic aspects of apical zonation in the angiosperm pollen tube. Sex Plant Reprod 3:187–194CrossRefGoogle Scholar
  18. Holzinger A, Kwok EY, Hanson MR (2008) Effects of arc3, arc5 and arc6 mutations on plastid morphology and stromule formation in green and non-green tissues of Arabidopsis thaliana. Photochem Photobiol 84:1324–1335CrossRefPubMedGoogle Scholar
  19. Inaba T, Alvarez-Huerta M, Li M, Bauer J, Ewers C, Kessler F, Schnell DJ (2005) Arabidopsis Tic110 is essential for the assembly and function of the protein import machinery of plastids. Plant Cell 17:1482–1496CrossRefPubMedGoogle Scholar
  20. Itoh R, Fujiwara M, Nagata N, Yoshida S (2001) A chloroplast protein homologous to the eubacterial topological specificity factor MinE plays a role in chloroplast division. Plant Physiol 127:1644–1655CrossRefPubMedGoogle Scholar
  21. Kanamaru K, Fujiwara M, Kim M, Nagashima A, Nakazato E, Tanaka K, Takahashi H (2000) Chloroplast targeting, distribution and transcriptional fluctuation of AtMinD1, a eubacteria-type factor critical for chloroplast division. Plant Cell Physiol 41:1119–1128CrossRefPubMedGoogle Scholar
  22. Kawai-Toyooka H, Kuramoto C, Orui K, Motoyama K, Kikuchi K, Kanegae T, Wada M (2004) DNA interference: a simple and efficient gene-silencing system for high-throughput functional analysis in the fern Adiantum. Plant Cell Physiol 45:1648–1657CrossRefPubMedGoogle Scholar
  23. Kirk JTO, Tilney-Bassett RAE (1978) The plastids. Elsevier/North-Holland, Amsterdam, The NetherlandsGoogle Scholar
  24. Köhler RH, Hanson MR (2000) Plastid tubules of higher plants are tissue-specific and developmentally regulated. J Cell Sci 113:81–89PubMedGoogle Scholar
  25. Kojo KH, Fujiwara MT, Itoh RD (2009) Involvement of AtMinE1 in plastid morphogenesis in various tissues of Arabidopsis thaliana. Biosci Biotechnol Biochem 73:2632–2639CrossRefPubMedGoogle Scholar
  26. Kuang A, Musgrave ME (1996) Dynamics of vegetative cytoplasm during generative cell formation and pollen maturation in Arabidopsis thaliana. Protoplasma 194:81–90CrossRefPubMedGoogle Scholar
  27. Kuroiwa T, Kuroiwa H, Sakai A, Takahashi H, Toda K, Itoh R (1998) The division apparatus of plastids and mitochondria. Int Rev Cytol 181:1–41CrossRefPubMedGoogle Scholar
  28. López-Juez E, Pyke KA (2005) Plastids unleashed: their development and their integration in plant development. Int J Dev Biol 49:557–577CrossRefPubMedGoogle Scholar
  29. Lovy-Wheeler A, Cárdenas L, Kunkel JG, Hepler PK (2007) Differential organelle movement on the actin cytoskeleton in lily pollen tubes. Cell Motil Cytoskeleton 64:217–232CrossRefPubMedGoogle Scholar
  30. Maple J, Møller SG (2007) Plastid division coordination across a double-membraned structure. FEBS Lett 581:2162–2167CrossRefPubMedGoogle Scholar
  31. Marrison JL, Rutherford SM, Robertson EJ, Lister C, Dean C, Leech RM (1999) The distinctive roles of five different ARC genes in the chloroplast division process in Arabidopsis. Plant J 18:651–662CrossRefPubMedGoogle Scholar
  32. Matsushima R, Hamamura Y, Higashiyama T, Arimura S, Sodmergen TN, Sakamoto W (2008) Mitochondrial dynamics in plant male gametophyte visualized by fluorescent live imaging. Plant Cell Physiol 49:1074–1083CrossRefPubMedGoogle Scholar
  33. Miyagishima S, Nishida K, Mori T, Matsuzaki M, Higashiyama T, Kuroiwa H, Kuroiwa T (2003) A plant-specific dynamin-related protein forms a ring at the chloroplast division site. Plant Cell 15:655–665CrossRefPubMedGoogle Scholar
  34. Miyagishima S, Nozaki H, Nishida K, Nishida K, Matsuzaki M, Kuroiwa T (2004) Two types of FtsZ proteins in mitochondria and red-lineage chloroplasts: the duplication of FtsZ is implicated in endosymbiosis. J Mol Evol 58:291–303CrossRefPubMedGoogle Scholar
  35. Mori T, Tanaka I (2000) Isolation of ftsZ gene from plastid-deficient generative cells of Lilium longiflorum. Protoplasma 214:57–64CrossRefGoogle Scholar
  36. Mori T, Takahara M, Miyagishima S, Kuroiwa H, Kuroiwa T (2001) Visualization of FtsZ rings in plastids of the microspore in Lilium longiflorum. Cytologia 66:113–115Google Scholar
  37. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue culture. Physiol Plant 103:473–497CrossRefGoogle Scholar
  38. Nagata N, Saito C, Sakai A, Kuroiwa H, Kuroiwa T (1999) The selective increase or decrease of organellar DNA in generative cells just after pollen mitosis one controls cytoplasmic inheritance. Planta 209:53–65CrossRefPubMedGoogle Scholar
  39. Nakanishi H, Suzuki K, Kabeya Y, Miyagishima S (2009) Plant-specific protein MCD1 determines the site of chloroplast division in concert with bacteria-derived MinD. Curr Biol 19:151–156CrossRefPubMedGoogle Scholar
  40. Okazaki K, Kabeya Y, Suzuki K, Mori T, Ichikawa T, Matsui M, Nakanishi H, Miyagishima S (2009) The PLASTID DIVISION1 and 2 components of the chloroplast division machinery determine the rate of chloroplast division in land plant cell differentiation. Plant Cell 21:1769–1780CrossRefPubMedGoogle Scholar
  41. Osteryoung KW, Stokes KD, Rutherford SM, Percival AL, Lee WY (1998) Chloroplast division in higher plants requires members of two functionally divergent gene families with homology to bacterial ftsZ. Plant Cell 10:1991–2004CrossRefPubMedGoogle Scholar
  42. Pacini E (1996) Types and meaning of pollen carbohydrate reserves. Sex Plant Reprod 9:362–366CrossRefGoogle Scholar
  43. Possingham JV, Lawrence ME (1983) Controls to plastid division. Int Rev Cytol 84:1–56CrossRefGoogle Scholar
  44. Primavesi LF, Wu H, Mudd EA, Day A, Jones HD (2007) Visualisation of plastids in endosperm, pollen and root plastids of transgenic wheat expressing modified GFP fused to transit peptides from wheat SSU RubisCO, rice FtsZ and maize ferredoxin III proteins. Transgenic Res 17:529–543CrossRefPubMedGoogle Scholar
  45. Pyke KA, Page AM (1998) Plastid ontogeny during petal development in Arabidopsis. Plant Physiol 116:797–803CrossRefPubMedGoogle Scholar
  46. Robertson EJ, Pyke KA, Leech RM (1995) arc6, an extreme chloroplast division mutant of Arabidopsis also alters proplastid proliferation and morphology in shoot and root apices. J Cell Sci 108:2937–2944PubMedGoogle Scholar
  47. Rothfield L, Taghbalout A, Shih YL (2005) Spatial control of bacterial division-site placement. Nat Rev Microbiol 3:959–968CrossRefPubMedGoogle Scholar
  48. Rotman N, Durbarry A, Wardle A, Yang WC, Chaboud A, Faure JE, Berger F, Twell D (2005) A novel class of MYB factors controls sperm-cell formation in plants. Curr Biol 15:244–248CrossRefPubMedGoogle Scholar
  49. Rutherford G, Tanurdzic M, Hasebe M, Banks JA (2004) A systemic gene silencing method suitable for high throughput, reverse genetic analyses of gene function in fern gametophytes. BMC Plant Biol 4:6CrossRefPubMedGoogle Scholar
  50. Shimmen T, Yokota E (2004) Cytoplasmic streaming in plants. Curr Opin Cell Biol 16:68–72CrossRefPubMedGoogle Scholar
  51. Smyth DR, Bowman JL, Meyerowitz EM (1990) Early flower development in Arabidopsis. Plant Cell 2:755–767CrossRefPubMedGoogle Scholar
  52. Spurr AR (1969) A low-viscosity resin embedding medium for electron microscopy. J Ultrastruct Res 26:31–34CrossRefPubMedGoogle Scholar
  53. Stokes KD, McAndrew RS, Figueroa R, Vitha S, Osteryoung KW (2000) Chloroplast division and morphology are differentially affected by overexpression of FtsZ1 and FtsZ2 genes in Arabidopsis. Plant Physiol 124:1668–1677CrossRefPubMedGoogle Scholar
  54. Strepp R, Scholz S, Kruse S, Speth V, Reski R (1998) Plant nuclear gene knockout reveals a role in plastid division for the homolog of the bacterial cell division protein FtsZ, an ancestral tubulin. Proc Natl Acad Sci USA 95:4368–4373CrossRefPubMedGoogle Scholar
  55. Tanaka I (1991) Microtubule-determined plastid distribution during microsporogenesis in Lilium longiflorum. J Cell Sci 99:21–31Google Scholar
  56. Tang LY, Nagata N, Matsushima R, Chen Y, Yoshioka Y, Sakamoto W (2009) Visualization of plastids in pollen grains: involvement of FtsZ1 in pollen plastid division. Plant Cell Physiol 50:904–908CrossRefPubMedGoogle Scholar
  57. Vitha S, McAndrew RS, Osteryoung KW (2001) FtsZ ring formation at the chloroplast division site in plants. J Cell Biol 153:111–119CrossRefPubMedGoogle Scholar
  58. Wang Z, Pesacreta TC (2004) A subclass of myosin XI is associated with mitochondria, plastids, and the molecular chaperone subunit TCP-1α in maize. Cell Motil Cytoskeleton 57:218–232CrossRefPubMedGoogle Scholar
  59. 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:e718CrossRefPubMedGoogle Scholar
  60. Yamaoka S, Leaver CJ (2008) EMB2473/MIRO1, an Arabidopsis Miro GTPase, is required for embryogenesis and influences mitochondrial morphology in pollen. Plant Cell 20:589–601CrossRefPubMedGoogle Scholar
  61. Yang Y, Glynn JM, Olson BJSC, Schmitz AJ, Osteryoung KW (2008) Plastid division: across time and space. Curr Opin Plant Biol 11:1–8CrossRefGoogle Scholar
  62. Yoder DW, Kadirjan-Kalbach D, Olson BJ, Miyagishima S, Deblasio SL, Hangarter RP, Osteryoung KW (2007) Effects of mutations in Arabidopsis FtsZ1 on plastid division, FtsZ ring formation and positioning, and FtsZ filament morphology in vivo. Plant Cell Physiol 48:775–791CrossRefPubMedGoogle Scholar
  63. Yoshida Y, Kuroiwa H, Misumi O, Nishida K, Yagisawa F, Fujiwara T, Nanamiya H, Kawamura F, Kuroiwa T (2006) Isolated chloroplast division machinery can actively constrict after stretching. Science 313:1435–1438CrossRefPubMedGoogle Scholar
  64. Zheng M, Wang Q, Teng Y, Wang X, Wang F, Chen T, Samaj J, Lin J, Logan DC (2010) The speed of mitochondrial movement is regulated by the cytoskeleton and myosin in Picea wilsonii pollen tubes. Planta 231:779–791CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Makoto T. Fujiwara
    • 1
    • 2
    Email author
  • Haruki Hashimoto
    • 2
  • Yusuke Kazama
    • 1
  • Tomonari Hirano
    • 1
  • Yasushi Yoshioka
    • 3
  • Seishiro Aoki
    • 2
  • Naoki Sato
    • 2
  • Ryuuichi D. Itoh
    • 4
  • Tomoko Abe
    • 1
  1. 1.RIKEN Nishina CenterSaitamaJapan
  2. 2.Department of Life Sciences, Graduate School of Arts and SciencesUniversity of TokyoTokyoJapan
  3. 3.Division of Biological Science, Graduate School of ScienceNagoya UniversityNagoyaJapan
  4. 4.Department of Chemistry, Biology and Marine Science, Faculty of ScienceUniversity of the RyukyusOkinawaJapan

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