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

Klaas Wulfetange,Sergey N Lomin,Georgy A Romanov,Andrea Stolz,Alexander Heyl,Thomas Schmülling

doi:10.1104/pp.111.180539

Klaas Wulfetange, Sergey N Lomin + Show 4 more

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https://doi.org/10.1104/pp.111.180539

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Journal: Plant Physiology	Publication Date: Aug 5, 2011
Citations: 197	License type: CC BY 4.0

Affiliation: Freie Universität Berlin

Abstract

The plant hormone cytokinin is perceived by membrane-located sensor histidine kinases. Arabidopsis (Arabidopsis thaliana) possesses three cytokinin receptors: ARABIDOPSIS HISTIDINE KINASE2 (AHK2), AHK3, and CYTOKININ RESPONSE1/AHK4. The current model predicts perception of the cytokinin signal at the plasma membrane. However, cytokinin-binding studies with membrane fractions separated by two-phase partitioning showed that in the wild type, as well as in mutants retaining only single cytokinin receptors, the major part of specific cytokinin binding was associated with endomembranes. Leaf epidermal cells of tobacco (Nicotiana benthamiana) expressing receptor-green fluorescent protein fusion proteins and bimolecular fluorescence complementation analysis showed strong fluorescence of the endoplasmic reticulum (ER) network for all three receptors. Furthermore, separation of the microsomal fraction of Arabidopsis plants expressing Myc-tagged AHK2 and AHK3 receptors by sucrose gradient centrifugation followed by immunoblotting displayed the Mg²⁺-dependent density shift typical of ER membrane proteins. Cytokinin-binding assays, fluorescent fusion proteins, and biochemical fractionation all showed that the large majority of cytokinin receptors are localized to the ER, suggesting a central role of this compartment in cytokinin signaling. A modified model for cytokinin signaling is proposed.

Highlights

The plant hormone cytokinin is perceived by membrane-located sensor histidine kinases
Cytokinin Receptor Localization receptors are localized to the plasma membrane (PM), which was initially based on bioinformatic analysis of the protein sequence and analogy with sensor His kinase localization in bacteria and yeast (Inoue et al, 2001; Ueguchi et al, 2001)
An in vitro cytokinin-binding assay with microsomes showed [3H]trans-zeatin-specific binding in both membrane fractions (Fig. 1, B–G). Quantitative analysis of this binding in wild-type plants revealed a highaffinity interaction with an equilibrium dissociation constant (KD) in the low nanomolar range (Fig. 1B). This is similar to the KD for trans-zeatin-receptor complexes of AHK3 and CYTOKININ RESPONSE1 (CRE1)/AHK4 (Yamada et al, 2001; Romanov et al, 2005, 2006) and the AHK2CHASE-TM domain (Stolz et al, 2011) expressed in bacteria

Summary

Introduction

The plant hormone cytokinin is perceived by membrane-located sensor histidine kinases. Cytokinins are a class of phytohormones regulating such diverse processes as root and shoot growth, leaf senescence, and chloroplast development They function in mediating the plant response to environmental signals, such as biotic and abiotic stresses (Argueso et al, 2009; Werner and Schmulling, 2009). Cytokinin Receptor Localization receptors are localized to the plasma membrane (PM), which was initially based on bioinformatic analysis of the protein sequence and analogy with sensor His kinase localization in bacteria and yeast (Inoue et al, 2001; Ueguchi et al, 2001). This assignment was experimentally supported by the localization of an overexpressed AHK3-GFP fusion protein at the PM of Arabidopsis protoplasts (Kim et al, 2006).

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