Studies of cytokinin receptor–phosphotransmitter interaction provide evidences for the initiation of cytokinin signalling in the endoplasmic reticulum

Abstract

Cytokinin receptors were shown recently to be localised mainly to the endoplasmic reticulum (ER); however, the activity of ER-located receptors was not proven. We have therefore tested the functionality of ER-located Arabidopsis receptors. The first step of cytokinin signal transduction is the transfer of a phosphoryl group from the activated receptor to a phosphotransfer protein. To determine the subcellular localisation of receptor–phosphotransmitter interaction in planta, BiFC experiments were performed. Receptors ARABIDOPSIS HISTIDINE KINASE 2 (AHK2), AHK3 and AHK4 (CRE1) and phosphotransmitters ARABIDOPSIS HISTIDINE-CONTAINING PHOSPHOTRANSMITTER 1 (AHP1), AHP2 and AHP3 fused to split-eYFP were transiently expressed in Nicotiana benthamiana leaves. Receptor–phosphotransmitter pairs were shown to interact in every possible combination in a pattern reflecting the ER. Receptor dimers, an active form of the receptors, were also detected in the ER. According to BiFC and protease protection data, the catalytic part of AHK3 was located in the cytoplasm whereas the hormone binding module faced the ER lumen. This topology is consistent with receptor signalling from the ER membrane. Finally, the functionality of receptors in different membrane fractions was tested using an in vitro kinase assay visualising the phosphorylation of phosphotransfer proteins. The detected cytokinin-dependent phosphotransfer activity was confined mainly to the ER-enriched fraction. Collectively, our data demonstrate that ER-located cytokinin receptors are active in cytokinin signal transduction. Hence, intracellular cytokinins appear to play an essential role in cytokinin signalling. An updated model for the spatial organisation of cytokinin transport form activation, intracellular trafficking and signalling from the ER is proposed.

Cytokinin signaling: from the ER or from the PM? That is the question!

“…Therefore, the subcellular site of interaction between functionally active receptors with phosphotransfer proteins would indicate the site of cytokinin signaling initiation. The subcellular localization of receptor–phosphotransmitter interaction in planta and the phosphoryl‐transmitting activity of cytokinin receptors located in plant membranes (total or fractionated into different membrane types) was reported recently (Lomin et al ., ). This analysis yielded support for receptor functioning in the ER.…”

“…Finally, there are several potential advantages in the intracellular localization of cytokinin receptors. ER location of the receptors includes the perinuclear space where they have been detected both as single fluorescent labeled protein (Caesar et al ., ; Wulfetange et al ., ) as well as part of a complex with phosphotransmitters and/or other receptors (forming homo‐ and heterodimers) in BiFC assays (Lomin et al ., ). Because of the shorter distance to the nucleus, signaling from these receptors would increase the velocity and the reliability of cytokinin signal transduction.…”

“…In the case of ER‐localized ethylene receptors it has been argued that receptor localization in the ER is energetically more efficient, because the receptor is not exported all the way through the secretory system to the PM, and receptors are more rapidly delivered to their site of action (Chen et al ., ). Finally, the ER‐localization of the receptor offers an opportunity for cytokinin signaling regulation and crosstalk also at the level of intracellular hormone trafficking and metabolic conversion (Lomin et al ., ).…”

“…Based on the data reported above it is undisputed that the vast majority of the cytokinin receptors is located in the ER membrane, at least in some tissues of Arabidopsis and maize (Caesar et al ., ; Lomin et al ., , ; Wulfetange et al ., ). Recent data obtained with cytokinin receptors from rice and apple tree confirmed their ER localization (Daudu et al ., ; Ding et al ., ).…”

“…The vast majority of the receptors is located there and fulfills a number of requirements that are needed for active signaling. This includes the correct topology, the specific binding of cytokinin at a pH typical for this cellular compartment, the interaction with phosphotransfer proteins and the transfer of the phosphoryl group from ATP to phosphotransfer proteins in a cytokinin‐dependent manner (Lomin et al ., , , ; Wulfetange et al ., ). This has been studied most extensively in Arabidopsis leaf cells where AHK3 has a dominant role.…”