Biochemical characteristics and ligand-binding properties of Arabidopsis cytokinin receptor AHK3 compared to CRE1/AHK4 as revealed by a direct binding assay.
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 K(D) of 1-2 nM for the AHK3 receptor-hormone complex, which is close to the K(D) (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 (3)H-zeatin, and influenced by monovalent cations, while divalent cations (Ca(2+), Mg(2+), Mn(2+)) 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.
Romanov
Georgy A
GA
Free University of Berlin, Institute of Biology/Applied Genetics, Albrecht-Thaer-Weg 6, D-14195 Berlin, Germany. gar@appras.ru
Lomin
Sergey N
SN
Schmülling
Thomas
T
D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D001667:Binding, Competitive; D003583:Cytokinins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D000071677:Histidine Kinase; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D030781:Organisms, Genetically Modified; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D015026:Zeatin / Q000378:metabolism
D029681:Arabidopsis Proteins; D003583:Cytokinins; D008024:Ligands; D011956:Receptors, Cell Surface; D015026:Zeatin; D011494:Protein Kinases; D000071677:Histidine Kinase; C000605493:AHK3 protein, Arabidopsis; C421783:WOL protein, Arabidopsis