Detection of membrane‐bound cytokinin‐binding proteins in Arabidopsis thaliana cells

Abstract

In order to isolate cytokinin-binding proteins (CBPs), we have developed new affinity probes constituted of a cytokinin such as zeatin riboside ([9R]Z) conjugated to a carrier protein. These probes were used for detecting CBPs in an ELISA procedure. The efficiency of the cytokinin conjugate in detecting CBPs was controlled with protein model: proteins having an affinity for cytokinin such as the monoclonal anti-[9R]Z antibodies did bind the cytokinin conjugate whereas proteins unable to bind cytokinin such as bovine serum albumin did not. Using these new affinity probes, we showed that CBPs are present in the membrane fraction of in vitro cultured Arabidopsis thaliana cells. The nature of the protein at the detected binding sites was demonstrated by submitting the microsomal proteins to a proteolytic treatment, which was found to eradicate the binding. Free biologically active cytokinins or monoclonal anti-[9R]Z antibodies inhibited the binding, thus showing the specificity of the interaction. The detected CBPs were partially solubilized from the membranes with potassium chloride, indicating their peripheral membrane location. The separation by anion exchange chromatography of solubilized microsomal proteins revealed the existence of two different CBPs. They were present at higher levels in cells during the exponential growth phase.

The Cytokinin Receptors of Arabidopsis Are Located Mainly to the Endoplasmic Reticulum

“…The ER localization is consistent with the biochemical properties of cytokinin receptors (Romanov et al, 2006) and with earlier studies that identified highaffinity cytokinin-binding proteins in particulate/microsomal fractions of plant cells (Gardner et al, 1978;Kobayashi et al, 1981;Brault et al, 1999). The ER localization of the cytokinin receptors contrasts with the previous prediction that the cytokinin-binding CHASE domain is apoplastic (Inoue et al, 2001;Ueguchi et al, 2001) and with current models of cytokinin signaling assuming the localization of cytokinin receptors at the PM (Heyl and Schmü lling, 2003;Hwang and Sakakibara, 2006;Mü ller and Sheen, 2007;To and Kieber, 2008).…”

Purification and Identification of a 42-Kilodalton Abscisic Acid-Specific-Binding Protein from Epidermis of Broad Bean Leaves

“…The cold acetone washing, ammonium sulfate precipitation, and Triton X-100 washing were often used in the extraction of plant hormone-binding proteins (Shimomura et al, 1986;Sugaya and Sakai, 1996;Brault et al, 1997Brault et al, , 1999, among which we adopted Triton X-100 as an unique washing medium because both the cold acetone washing and ammonium sulfate precipitation were shown to reduce or even to abolish the ABA-binding activity. This may be because the ABA-binding proteins are generally membrane-bound proteins (Hocking et al, 1978;Hornberg and Weiler, 1984;Anderson et al, 1994;MacRobbie, 1995;Pedron et al, 1998; Zhang et al, Figure 9.…”

Cytokinins - recent news and views of evolutionally old molecules

“…Several authors report on biochemical approaches leading to identification of CKbinding proteins (CBPs) in several plant species such as maize, barley, wheat, oats (Avena sativa L.), tobacco, cucumber (Cucumis sativus L.), carrot (Daucus carota L.) and Vigna radiata (L.) R. Wilczek (Brinegar 1994;Brault et al 1999;Kamínek et al 2003;Pasternak et al 2006). Despite the many CBPs that exhibited binding of CKs in vitro, their K d values were too low to qualify them as receptors.…”