JXB Advance Access originally published online on October 30, 2006
Journal of Experimental Botany 2006 57(15):4051-4058; doi:10.1093/jxb/erl179
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RESEARCH PAPER |
Biochemical characteristics and ligand-binding properties of Arabidopsis cytokinin receptor AHK3 compared to CRE1/AHK4 as revealed by a direct binding assay
1Free University of Berlin, Institute of Biology/Applied Genetics, Albrecht-Thaer-Weg 6, D-14195 Berlin, Germany
2Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya 35, 127276 Moscow, Russia
* To whom correspondence should be addressed. E-mail: gar{at}ippras.ru or gromanov{at}yahoo.com
The cytokinin receptor AHK3 of Arabidopsis thaliana plays a predominant role in shoot development. A study of the hormone-binding characteristics of AHK3 compared with the mainly root-confined receptor CRE1/AHK4 has been accomplished using a live-cell binding assay on transgenic bacteria expressing individual receptor proteins. Both receptors bound trans-zeatin (tZ) with high affinity. Scatchard analysis showed a linear function corresponding to an apparent KD of 1–2 nM for the AHK3 receptor–hormone complex, which is close to the KD (2–4 nM) for the CRE1/AHK4 receptor–hormone complex. The specific binding of tZ to both receptors was pH dependent, AHK3 being more sensitive to pH changes than CRE1/AHK4. Hormone binding was reversible, at least for the bulk of 3H-zeatin, and influenced by monovalent cations, while divalent cations (Ca2+, Mg2+, Mn2+) at physiological concentrations had no significant effect. AHK3 differed significantly from CRE1/AHK4 in relative affinity to some cytokinins. AHK3 had an approximately 10-fold lower affinity to isopentenyladenine (iP) and its riboside, but a higher affinity to dihydrozeatin than CRE1/AHK4. For AHK3, cytokinin ribosides (tZR, iPR) and cis-zeatin had true binding activity, although lower than that of tZ. The phenylurea-derived cytokinin thidiazuron was a strong competitor and bound to the same site as did adenine-derived cytokinins. The inhibitor of cytokinin action butan-1-ol had little effect on cytokinin–receptor complex formation. The revealed properties of AHK3 suggest its specific function in root-to-shoot communication.
Key words: AHK3, Arabidopsis thaliana, binding assay, CRE1/AHK4, cytokinin, hormone–receptor complex, receptor, sensor histidine kinase, zeatin
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