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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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