Advertisement

Fast loading ester fluorescent Ca2+ and pH indicators into pollen of Pyrus pyrifolia

  • 730 Accesses

  • 14 Citations

Abstract

Loading of Ca2+-sensitive fluorescent probes into plant cells is an essential step to measure activities of free Ca2+ ions in cytoplasm with a fluorescent imaging technique. Fluo-3 is one of the most suitable Ca2+ indicators for CLSM. We loaded pollen with fluo-3/AM at three different temperatures. Fluo-3/AM was successfully loaded into pollen at both low (4°C) and high (37°C) temperatures. However, high loading temperature was best suited for pollen, because germination rate of pollen and growth of pollen tubes were relatively little impaired and loading time was shortened. Moreover, Ca2+ distribution increased in the three apertures of pollen after hydration and showed a Ca2+ gradient, similar to the tip of growing pollen tubes. The same protocol can be used with the AM-forms of other fluorescent dyes for effective labeling. When loading BCECF-AM into pollen at high temperature, the pollen did not show a pH gradient after hydration. Ca2+ activities and fluxes had the same periodicity as pollen germination, but pH did not show the same phase and mostly lagged behind. However, the clear zone was alkaline when pollen tube growth was slowed or stopped and turned acidic when growth recovered. It is likely that apical pHi regulated pollen tube growth.

This is a preview of subscription content, log in to check access.

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 99

This is the net price. Taxes to be calculated in checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Abbreviations

BCECF-AM:

3′-O-Acetyl-2′,7′-bis(carboxyethyl)-4 or 5-carboxyfluorescein diacetoxymethyl ester

CLSM:

Confocal laser scanning microscopy

[Ca2+]cyt :

Cytosolic-free calcium

dd water:

Double distilled water

ECFP:

Enhanced cyan fluorescent protein

EGTA:

Ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid

EYFP:

Enhanced yellow fluorescent protein

pHi :

Intracellular pH

SSR:

Shortest significance ranges

References

  1. Bingquan W, Zheng J (2003) Primary functional identification of gene TMSG-1. Sci China C Life Sci 46:641–650

  2. Bush DS (1995) Calcium regulation in plant cells and its role in signalling. Annu Rev Plant Physiol Plant Mol Biol 46:95–122

  3. Bush D, Hedrich R, Schroeder J, Jones R (1988) Channel-mediated potassium ion flux in barley aleurone protoplasts. Planta 176:368–377

  4. Camacho L, Parton R, Trewavas A, Malhó R (2000) Imaging cytosolic free-calcium distribution and oscillations in pollen tubes with confocal microscopy: a comparison of different dyes and loading methods. Protoplasma 212:162–173

  5. Campanoni P, Blatt MR (2007) Membrane trafficking and polar growth in root hairs and pollen tubes. J Exp Bot 58:65–74

  6. Cao X-G, Li X–X, Bao Y-Z, Xing N-Z, Chen Y (2007) Responses of human lens epithelial cells to quercetin and DMSO. Investig Ophthalmol Vis Sci 48:3714–3719

  7. Cheung A, Wu H (2001) Pollen tube guidance—right on target. Science 293:1441–1442

  8. Cheung A, Wu H (2007) Structural and functional compartmentalization in pollen tubes. J Exp Bot 58:75–82

  9. Cody SH, Dubbin PN, Beischer A, Duncan ND, Hill JS, Kaye A, Williams DA (1993) Intracellular pH mapping with SNARF-1 and confocal microscopy. Micron 24:573–580

  10. Cork RJ (1986) Problems with the application of quin-2/AM to measuring cytoplasmic free calcium in plant cells. Plant Cell Environ 9:157–160

  11. Digonnet C, Aldon D, Leduc N, Dumas C, Rougier M (1997) First evidence of a calcium transient in flowering plants at fertilization. Development 124:2867–2874

  12. Feijó J, Sainhas J, Hackett G, Kunkel J, Hepler P (1999) Growing pollen tubes possess a constitutive alkaline band in the clear zone and a growth-dependent acidic tip. J Cell Biol 144:483–496

  13. Franklin-Tong V, Ride J, Read N, Trewavas A, Franklin F (1993) The self-incompatibility response in Paparer rhoeas is mediated by cytosolic-free calcium. Plant J 4:163–177

  14. Gadella TWJ, van der Krogt GNM, Bisseling T (1999) GFP-based FRET microscopy in living plant cells. Trends Plant Sci 4:287–291

  15. Ge LL, Tian HQ, Russell SD (2007) Calcium function and distribution during fertilization in angiosperms. Am J Bot 94:1046–1060

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

  17. Hepler P, Bosch M, Cardenas L, Lovy-Wheeler A, McKenna S, Wilsen K, Kunkel J (2005) Oscillatory pollen tube growth: Imaging the underlying structures and physiological processes. Microsc Microanal 11:148–149

  18. Hepler P, Lovy-Wheeler A, McKenna S, Kunkel J (2006) Ions and pollen tube growth. Plant Cell Monogr 3:47–69

  19. Holdaway-Clarke T, Weddle N, Kim S, Robi A, Parris C, Kunkel J, Hepler P (2003) Effect of extracellular calcium, pH and borate on growth oscillations in Lilium formosanum pollen tubes. J Exp Bot 54:65–72

  20. Iwano M, Shiba H, Miwa T, Che F-S, Takayama S, Nagai T, Miyawaki A, Isogai A (2004) Ca2+ dynamics in a pollen grain and papilla cell during pollination of Arabidopsis. Plant Physiol 136:3562–3571

  21. Jacobs WR, Mandel LJ (1987) Fluorescent measurements of intracellular free calcium in isolated toad urinary bladder epithelial cells. J Membr Biol 97:53–62

  22. Kajiura M, Kanato K, Machida Y, Maeda M, Kozaki I, Tashiro T, Kishimoto O, Seike K (1974) New Japanese pear cultivar ‘Hakko’ and ‘Hosui’. Bull Fruit Tree Res Stn 1:1–12

  23. Kao JPY (1994) Practical aspects of measuring [Ca2+] with fluorescent indicators. Methods Cell Biol 40:155–181

  24. Kao J, Harootunian A, Tsien R (1989) Photochemically generated cytosolic calcium pulses and their detection by fluo-3. J Biol Chem 264:8179–8184

  25. Kuchitsu K, Ward J, Allen G, Schelle I, Schroeder J (2002) Loading acetoxymethyl ester fluorescent dyes into the cytoplasm of Arabidopsis and Commelina guard cells. New Phytol 153:527–533

  26. Malhó R, Read N, Pais M, Trewavas A (1994) Role of cytosolic free calcium in the reorientation of pollen tube growth. Plant J 5:331–341

  27. Marches R, Vitetta E, Uhr J (2001) A role for intracellular pH in membrane IgM-mediated cell death of human B lymphomas. Proc Natl Acad Sci 98:3434–3439

  28. Michard E, Dias P, Feijo J (2008) Tobacco pollen tubes as cellular models for ion dynamics: improved spatial and temporal resolution of extracellular flux and free cytosolic concentration of calcium and protons using pHluorin and YC3. 1 CaMeleon. Sex Plant Reprod 21:169–181

  29. Minta A, Kao JP, Teien RY (1989) Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J Biol Chem 264:8171–8178

  30. Miyawaki A, Griesbeck O, Heim R, Tsien RY (1999) Dynamic and quantitative Ca2+ measurements using improved cameleons. Proc Natl Acad Sci 96:2135–2140

  31. Mól R, Filek M, Dumas C, Matthys-Rochon E (2004) Cytoplasmic calcium in silk trichomes after pollen grain reception and post-pollination changes of the electric potential in pistil tissues of maize. Plant Sci 166:1461–1469

  32. Monshausen G, Messerli M (2008) Imaging of the Yellow Cameleon 3.6 indicator reveals that elevations in cytosolic Ca2+ follow oscillating increases in growth in root hairs of Arabidopsis thaliana. Plant Physiol 147:1690–1698

  33. Ono K, Wang X, Han J (2001) Resistance to tumor necrosis factor-induced cell death mediated by PMCA4 deficiency. Mol Cell Biol 21:8276–8288

  34. Park Y, Smith RD, Combs AB, Kehrer JP (1988) Prevention of acetaminophen-induced hepatotoxicity by dimethyl sulfoxide. Toxicology 52:165–175

  35. Pierson ES, Miller DD, Callaham DA, Shipley AM, Rivers BA, Cresti M, Hepler PK (1994) Pollen tube growth is coupled to the extracellular calcium ion flux and the intracellular calcium gradient: effect of BAPTA-type buffers and hypertonic media. Plant Cell 6:1815–1828

  36. Pierson ES, Miller DD, Callaham DA, Van Aken J, Hackett G, Hepler PK (1996) Tip-localized calcium entry fluctuates during pollen tube growth. Dev Biol 174:160–173

  37. Qu H-Y, Shang Z-L, Zhang S-L, Liu L-M, Wu J-Y (2007) Identification of hyperpolarization-activated calcium channels in apical pollen tubes of Pyrus pyrifolia. New Phytol 174:524–536

  38. Rathore KS, Cork RJ, Robinson KR (1991) A cytoplasmic gradient of Ca2+ is correlated with the growth of lily pollen tubes. Dev Biol 148:612–619

  39. Rosa R, Sanfeliu C, Suol C, Pomés A, Rodríguez-Farré E, Schousboe A, Frandsen A (1997) The mechanism for hexachlorocyclohexane-induced cytotoxicity and changes in intracellular Ca2+ homeostasis in cultured cerebellar granule neurons is different for the α-and δ-isomers. Toxicol Appl Pharmacol 142:31–39

  40. Shang Z-l, Ma L-g, Zhang H-l, He R-r, Wang X-c, Cui S-j, Sun D-y (2005) Ca2+ Influx into lily pollen grains through a hyperpolarization-activated Ca2+-permeable channel which can be regulated by extracellular CaM. Plant Cell Physiol 46:598–608

  41. Spurr AR (1976) Low viscosity embedding medium. The Regents of the University of California, Berkeley

  42. Takahashi A, Camacho P, Lechleiter JD, Herman B (1999) Measurement of intracellular calcium. Am Physiol Soc 79:1089–1125

  43. Tian HQ, Kuang A, Musgrave ME, Russell SD (1998) Calcium distribution in fertile and sterile anthers of a photoperiod-sensitive genic male-sterile rice. Planta 204:183–192

  44. Tsien RY (1981) A non-disruptive technique for loading calcium buffers and indicators into cells. Nature 290:527–528

  45. Wang Y, Fan L, Zhang W, Zhang W, Wu W (2004) Ca2+-permeable channels in the plasma membrane of Arabidopsis pollen are regulated by actin microfilaments. Plant Physiol 136:3892–3904

  46. Watahiki MK, Trewavas AJ, Parton RM (2004) Fluctuations in the pollen tube tip-focused calcium gradient are not reflected in nuclear calcium level: a comparative analysis using recombinant yellow cameleon calcium reporter. Sex Plant Reprod 17:125–130

  47. Yamamoto Y, Nishimura M, Hara-Nishimura I, Noguchi T (2003) Behavior of vacuoles during microspore and pollen development in Arabidopsis thaliana. Plant Cell Physiol 44:1192–1201

  48. Zhang W-H, Rengel Z, Kuo J (1998) Determination of intracellular Ca2+ in cells of intact wheat roots: loading of acetoxymethyl ester of Fluo-3 under low temperature. Plant J 15:147–151

  49. Zhao Md, Hollingworth S, Baylor SM (1997) AM-loading of fluorescent Ca2+ indicators into intact single fibers of frog muscle. Biophys J 72:2736–2747

Download references

Acknowledgments

We thank Professor Megumi Iwano of Nara Institute of Science and Technology (Japan) who supplied transgenic seed that expressed the YC3.1 gene in pollen grains and papilla cells. We also thank Professor Deng Shaoping in ZheJiang GongShang University who let us freely use the confocal instrument in his laboratory. We thank all the scientists who helped to improve our manuscript. We are indebted to the two anonymous reviewers, whose insightful comments resulted in a much improved final version of the manuscript. This work was supported by the National Natural Science Foundation of China (31071759) and the Scientific Research Fund of Huaiyin Institute of Technology (No: HG0606).

Author information

Correspondence to Shaoling Zhang.

Electronic supplementary material

Below is the link to the electronic supplementary material.10265_2011_440_MOESM2_ESM.doc

There are three phases of movie of the same pollen tube. Pollen were cultured in dye-free medium for 2 h in darkness after loading BCECF-AM at high temperature (37°C) for 30 min and then observed with CLSM. Confocal images were collected every 10 min for 3 h. We adjusted field of vision of CLSM every hour, because of movement from pollen tube growth. The first movie (pH 1.mpg) is images collected during the first 0–50 min (mpg 525 kb)

The second movie (pH 2.mpg) is images collected during the middle 60–130 min (mpg 829 kb)

The third movie (pH 3.mpg) is images collected in the last 50 min. These movies reveal the change of pH dynamics at of pollen tube tips during their growth (mpg 423 kb)

There are three phases of movie of the same pollen tube. Pollen were cultured in dye-free medium for 2 h in darkness after loading BCECF-AM at high temperature (37°C) for 30 min and then observed with CLSM. Confocal images were collected every 10 min for 3 h. We adjusted field of vision of CLSM every hour, because of movement from pollen tube growth. The first movie (pH 1.mpg) is images collected during the first 0–50 min (mpg 525 kb)

The second movie (pH 2.mpg) is images collected during the middle 60–130 min (mpg 829 kb)

The third movie (pH 3.mpg) is images collected in the last 50 min. These movies reveal the change of pH dynamics at of pollen tube tips during their growth (mpg 423 kb)

Supplementary material 1 (DOC 103 kb)

Supplementary material 2 (DOC 10,543 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Qu, H., Jiang, X., Shi, Z. et al. Fast loading ester fluorescent Ca2+ and pH indicators into pollen of Pyrus pyrifolia . J Plant Res 125, 185–195 (2012). https://doi.org/10.1007/s10265-011-0440-z

Download citation

Keywords

  • CLSM
  • Pollen
  • Fluo-3/AM
  • Temperature
  • BCECF-AM