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Comparison of F-actin fluorescent labeling methods in pollen tubes of Lilium davidii

  • Cell Biology and Morphogenesis
  • Published:
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Abstract

Fluorescence labeling of F-actin in pollen tubes by various methods has produced inconsistent results in the literature. Here, we report that EGTA, which was always used in fixative buffers in the past and thought to help cytoskeleton stabilization, can significantly affect F-actin distribution and lead to the formation of thick F-actin bundles at the tip of the pollen tube. We also found that vacuum-infiltration for the first 5 min during pollen tube fixation can better preserve normal cytoplasm structure and F-actin distribution. In contrast, m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) treatment before chemical fixation resulted in a shortening of the free zone of thick F-actin bundles in the pollen tube tip. Taken together, our results suggest that exclusion of EGTA and MBS from the fixative buffer, in combination with vacuum-infiltration in the first 5 min of fixation, can improve F-actin fluorescence labeling in pollen tubes of Lilium davidii.

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Fig. 1a–d
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Abbreviations

FM:

Fixative method;

GFP:

Green fluorescent protein;

MBS:

m-Maleimidobenzoic acid N-hydroxysuccinimide ester

References

  • Baskin TI, Busby CH, Fowke LC, Sammut M, Gubler F (1992) Improvements in immunostaining samples embedded in methacrylate: localization of microtubules and other antigens throughout developing organs in plants of diverse taxa. Planta 187:405–413

    Article  Google Scholar 

  • Cai G, Moscatelli A, Cresti M (1997) Cytoskeletal organization and pollen tube growth. Trends Plant Sci 2:86–91

    Google Scholar 

  • Cramer LP, Briggs LJ, Dawe HR (2002) Use of fluorescently labelled deoxyribonuclease I to spatially measure G-actin levels in migrating and non-migrating cells. Cell Motil Cytoskeleton 51:27–38

    Google Scholar 

  • Doris FP, Steer MW (1996) Effects of fixatives and permeabilisation buffers on pollen tubes: implications for localization of actin microfilaments using phalloidin staining. Protoplasma 195:25–36

    Google Scholar 

  • Foissner I, Grolig F, Obermeyer G (2002) Reversible protein phosphorylation regulates the dynamic organization of the pollen tube cytoskeleton: effects of calyculin A and okadaic acid. Protoplasma 220:1–15

    Article  Google Scholar 

  • 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 152:1019–1032

    Article  Google Scholar 

  • Geitmann A, Emons AMC (2000) The cytoskeleton in plant and fungal cell tip growth. J Microsc 198:218–245

    Article  Google Scholar 

  • Geitmann A, Snowman BN, Emons AMC, Flanklin-Tong VE (2000) Alteration in the actin cytoskeleton of pollen tubes are induced by the self-incompatibility reaction in Papaver rhoeas. Plant Cell 12:1239–1251

    Google Scholar 

  • Gibbon BC, Kovar DR, Staiger CJ (1999) Latrunculin B has different effects on pollen germination and tube growth. Plant Cell 11:2349–2363

    Article  Google Scholar 

  • He Y, Wetzstein HY (1995) Fixation induces differential tip morphology and immunolocalization of the cytoskeleton in pollen tubes. Physiol Plant 93:757–763

    Article  Google Scholar 

  • Hepler PK (1997) Tip growth in pollen tubes: calcium leads the way. Trends Plant Sci 2:79–80

    Google Scholar 

  • Hepler PK, Vidali L, Cheung AY (2001) Polarized cell growth in higher plants. Annu Rev Cell Dev Biol 17:159–187

    Article  CAS  PubMed  Google Scholar 

  • Ketelaar T, de Ruijter NCA, Emons AMC (2003) Unstable F-actin specifies the area and microtubule direction of cell expansion in arabidopsis root hairs. Plant Cell 15:285–292

    Article  CAS  PubMed  Google Scholar 

  • Ketelaar T, Faivre-Moskalenko C, Esseling JJ, de Ruijter NCA, Grierson CS, Dogterom M, Emons AMC (2002) Positioning of nuclei in arabidopsis root hairs: an actin-regulated process of tip growth. Plant Cell 14:2941–2955

    Google Scholar 

  • Kost B, Spielhofer P, Chua NH (1998) A GFP-mouse talin fusion protein labels plant actin filaments in vivo and visualizes the actin cytoskeleton in growing pollen tubes. Plant J 16:393–401

    Article  CAS  PubMed  Google Scholar 

  • Kovar DR, Drøbak BK, Staiger CJ (2000) Maize profilin isoforms are functionally distinct. Plant Cell 12:583–598

    Google Scholar 

  • Li Y, Yan L-F, Zee S-Y (1998) Distribution of F-actin and microtubules in pollen and pollen tube of Lilium davidii. Acta Bot Sin 40:890–894

    Google Scholar 

  • Li Y, Zee SY, Liu YM, Huang BQ, Yen LF (2001) Circular F-actin bundles and a G-actin gradient in pollen and pollen tubes of Lilium davidii. Planta 213:722–730

    Article  Google Scholar 

  • Miller DD, de Ruijter NCA, Bisseling T, Emons AM (1999) The role of actin in root hair morphogenesis: studies with lipochito-oligosaccharide as a growth stimulator and cytochalasin as an actin perturbing drug. Plant J 17:141–154

    Article  CAS  Google Scholar 

  • Miller DD, Lancelle SA, Hepler PK (1996) Actin microfilaments do not form a dense meshwork in Lilium longiflorum pollen tube tips. Protoplasma 195:123–132

    Google Scholar 

  • Pierson ES (1988) Rhodamine-phalloidin staining of F-actin in pollen after dimethylsulphoxide permeabilization. Sex Plant Reprod 1:83–87

    Article  Google Scholar 

  • Smertenko AP, Allwood EG, Khan S, Jiang C-J, Maciver SK, Weeds AG, Hussey P (2001) Interaction of pollen-specific actin-depolymerizing factor with actin. Plant J 25:203–212

    Google Scholar 

  • Sonobe S, Shibaoka H (1989) Cortical fine actin filaments in higher plant cells visualized by rhodamine-phalloidin after pretreatment with m-maleimidobenzoyl N-hydroxysuccinimid estar. Protoplasma 148:80–86

    Google Scholar 

  • Staiger CJ (2000) Signaling to the actin cytoskeleton in plants. Annu Rev Plant Physiol Plant Mol Biol 51:257–288

    Google Scholar 

  • Sutoh K (1984) Actin-actin and actin-deoxyribonuclease I contact sites in the actin sequence. Biochemistry 23:1942–1946

    Google Scholar 

  • Taylor LP, Hepler PK (1997) Pollen germination and tube growth. Annu Rev Plant Physiol Plant Mol Biol 48:461–491

    Article  CAS  PubMed  Google Scholar 

  • Vidali L, Hepler PK (2001) Actin and pollen tube growth. Protoplasma 215:64–76

    Google Scholar 

  • Vidali L, McKenna ST, Hepler PK (2001) Actin polymerization is essential for pollen tube growth. Mol Biol Cell 12:2534–2545

    Google Scholar 

  • Vitha S, Baluška F, Braun M, Šamaj J, Volkmann D, Barlow PW (2000) Comparison of cryofixation and aldehyde fixation for plant actin immunocytochemistry: aldehydes do not destroy F-actin. Histochem J 32:457–466

    Google Scholar 

  • Wasteneys GO, Galway ME (2003) Remodeling the cytoskeleton for growth and form: an overview with some new views. Annu Rev Plant Biol 54:691–722

    Google Scholar 

  • Zheng Z-L, Yang Z (2000) The Rop GTPase switch turns on polar growth in pollen. Trends Plant Sci 5:298–303

    Google Scholar 

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Acknowledgements

We thank Ms. Shi-Wen Li for her help with the confocal microscope and the second reviewer for helpful comments. This study was supported by the National Natural Science Foundation of China (No. 30470109 and 30170458) to Yan Li

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Authors and Affiliations

  1. State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing, 100094, PR China

    Li Wang, Yi-Min Liu & Yan Li

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  1. Li Wang
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  2. Yi-Min Liu
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  3. Yan Li
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Correspondence to Yan Li.

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Communicated by H. Wang

Li Wang and Yi-Min Liu are considered joint first authors

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Wang, L., Liu, YM. & Li, Y. Comparison of F-actin fluorescent labeling methods in pollen tubes of Lilium davidii. Plant Cell Rep 24, 266–270 (2005). https://doi.org/10.1007/s00299-005-0935-y

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  • DOI: https://doi.org/10.1007/s00299-005-0935-y

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