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Preparation of rhodamine-labelled tubulin and its reassembly in guard cells in leaves of Vicia faba L.

Published online by Cambridge University Press:  12 February 2007

Yu Rong ,
Yuan Ming  and
Wang Xue-Chen
Yu Rong
Affiliation:
Department of Biology, Capital Normal University, Beijing 100037, China State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100094, China
Yuan Ming
Affiliation:
State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100094, China
Wang Xue-Chen*
Affiliation:
State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100094, China
*
*Corresponding author: E-mail: [email protected]
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Abstract

Tubulin from fresh pig brain was extracted through two cycles of polymerization and depolymerization, purified by chromatography on an ion-exchange column and labelled with 5(6)-carboxytetramethylrhodamine succinimidyl ester. We prepared 10.8 mg/ml rhodamine-labelled tubulin probe (dye/protein=0.6) and then microinjected it into living guard cells in leaves of Vicia faba L. After 5–10 min, a network of microtubules (MTs) could clearly be observed in the guard cells under a confocal laser scanning microscope. In opening guard cells, the cortical MTs radiated from the ventral wall to the dorsal wall and in closed guard cells the radial MTs were completely depolymerized into random and diffuse fragments. This shows that pig brain tubulin can easily be incorporated into MTs in guard cells, and that the dynamic behaviour of MTs can be examined directly in vivo during stomatal movement.

Keywords

rhodamine-labelled tubulinmicroinjectionguard cellsreassembly
Type
Research Article
Information
Chinese Journal of Agricultural Biotechnology , Volume 1 , Issue 3 , December 2004 , pp. 161 - 165
Copyright
Copyright © China Agricultural University and Cambridge University Press 2004

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References

Assmann, SM and Baskin, TI (1998) The function of guard cells does not require an intact array of cortical microtubules. Journal of Experimental Botany 49, 163–170.CrossRefGoogle Scholar
Bokros, CL, Hugdahl, JD and Hanesworth, VR et al. (1993) Characterization of the reversible taxol-induced polymerization of plant tubulin. Biochemistry 32, 3437–3447.Google Scholar
Cleary, AL (1995) F-actin redistributions at the division site in living Tradescantia stomatal complexes as revealed by microinjection of rhodamine-phalloidin. Protoplasma 185, 152–165.CrossRefGoogle Scholar
Hepler, PK, Cleary, AL and Gunning, BES et al. (1993) Cytoskeleton dynamics in living plant cells. Cell Biology International Reports 17, 127–142.Google Scholar
Huang, RF and Wang, XC (1997a) Roles of cytoplasmic microtubules in the regulation of stomatal movement. Acta Botanica Sinica 39, 253–258 in Chinese with English abstract.Google Scholar
Huang, RF and Wang, XC (1997b) Abscisic acid-induced changes in the orientation of cortical microtubules and their effects on stomatal movement of Vicia faba L. Acta Botanica Sinica 39, 375–378 in Chinese with English abstract.Google Scholar
Huang, RF, Wang, XC and Lou, CH (2000) Cytoskeletal inhibitors suppress the stomatal opening of Vicia faba L. induced by fusicoccin and IAA. Plant Science 156, 65–71.CrossRefGoogle ScholarPubMed
Hussey, PJ, Yuan, M and Calder, G et al. (1998) Microinjection of pollen-specific actin-depolymerizing factor, ZmADF1, reorientates F-actin strands in Tradescantia stamen hair cells. The Plant Journal 14, 353–357.Google Scholar
Kim, MJ, Hepler, PK and Eun, SO et al. (1995) Actin filaments in mature guard cells are radially distributed and involved in stomatal movement. Plant Physiology 109, 1077–1084.CrossRefGoogle ScholarPubMed
McAinsh, MR, Webb, AR and Taylor, JE et al. (1995) Stimulus-induced oscillations in guard cell cytosolic free calcium. Plant Cell 7, 1207–1219.Google Scholar
Marcus, AI, Moore, RC and Cyr, RC (2001) The role of microtubules in guard cell function. Plant Physiology 125, 387–395.Google Scholar
Morejohn, LC and Fosket, DE (1982) Higher plant tubulin identified by self-assembly into microtubules in vitro. Nature 297, 426–429.Google Scholar
Murphy, DB (1982) Assembly–disassembly purification and characterization of microtubule protein without glycerol. Methods in Cell Biology 24, 30–49.Google Scholar
Sloboda, RD, Dentler, WL and Rosenbaum, JL (1976) Microtubule-associated proteins and the stimulation of tubulin assembly in vitro. Biochemistry 15, 4497–4505.Google Scholar
Wasteneys, GO, Gunning, BES and Hepler, PK (1993) Microinjection of fluorescent brain tubulin reveals dynamic properties of cortical microtubules in living plant cells. Cell Motility and the Cytoskeleton 24, 205–213.CrossRefGoogle Scholar
Yu, R, Huang, RF and Wang, XC et al. (2001) Microtubule dynamics are involved in stomatal movement of Vicia faba L. Protoplasma 216, 113–118.Google Scholar
Yuan, M, Shaw, PJ and Warn, RM et al. (1994) Dynamic reorientation of cortical microtubules, from transverse to longitudinal, in living plant cells. Proceedings of National Academy of Sciences of the USA 91, 6050–6053CrossRefGoogle ScholarPubMed