Cytokinin fluoroprobe reveals multiple sites of cytokinin perception at plasma membrane and endoplasmic reticulum

Abstract

Plant hormone cytokinins are perceived by a subfamily of sensor histidine kinases (HKs), which via a two-component phosphorelay cascade activate transcriptional responses in the nucleus. Subcellular localization of the receptors proposed the endoplasmic reticulum (ER) membrane as a principal cytokinin perception site, while study of cytokinin transport pointed to the plasma membrane (PM)-mediated cytokinin signalling. Here, by detailed monitoring of subcellular localizations of the fluorescently labelled natural cytokinin probe and the receptor ARABIDOPSIS HISTIDINE KINASE 4 (CRE1/AHK4) fused to GFP reporter, we show that pools of the ER-located cytokinin receptors can enter the secretory pathway and reach the PM in cells of the root apical meristem, and the cell plate of dividing meristematic cells. Brefeldin A (BFA) experiments revealed vesicular recycling of the receptor and its accumulation in BFA compartments. We provide a revised view on cytokinin signalling and the possibility of multiple sites of perception at PM and ER.

Cell-surface receptors enable perception of extracellular cytokinins

“…10). Similar to previous 11,12 and recent 28 reports, some of the non-ER AHK signal was clearly at the cell surface, indicating plasma membrane localisation. Across multiple imaged cells, 25% of AHK3 and 36% of AHK4 signals were localised in non-ER regions (Fig.…”

“…Although we did not attempt to make detailed quantitation of relative proportions of AHKs on plasma membrane vs ER, our image analysis likewise clearly indicates that significant amounts of AHK are found in non-ER regions including the cell surface. Our data are strongly corroborated by independent experiments showing colocalisation of plasma membrane markers both with AHK4-GFP and with a fluorescently tagged iP derivative that preferentially binds AHK4 28 . Moreover, Kubiasová et al 28 show that AHK4 and tagged iP are found in brefeldin A-dependent endocytic vesicles characteristic of trafficking to and from the plasma membrane.…”

“…Our data are strongly corroborated by independent experiments showing colocalisation of plasma membrane markers both with AHK4-GFP and with a fluorescently tagged iP derivative that preferentially binds AHK4 28 . Moreover, Kubiasová et al 28 show that AHK4 and tagged iP are found in brefeldin A-dependent endocytic vesicles characteristic of trafficking to and from the plasma membrane. Despite the strong evidence for ER location of AHKs, recent reviews have highlighted the lack of direct substantiation of extracellular cytokinin perception 1,15,16 .…”

“…Cytokinin treatment was for 16 h then 1 μM FM4-64 applied 5 min prior to confocal imaging. See also Supplementary Figs cell, thus substantially resolving the lengthy debate on this issue 1,11,12,15,16,24,28,29 . Dual location of receptors may potentially provide plants with additional flexibility in cytokinin responses.…”

“…Given that tZ cytokinins have previously been suggested to be dispensable for root function 34 , our evidence for a dominant role of tZ rather than iP is surprising. Moreover, although Kubiasová et al 28 did not test tZ, they show that a tagged version of iP can enter the secretory pathway, resulting in delivery to the apoplast. One explanation for the discrepancy could be that endogenous and exogenous iP compounds behave differently, for example, if the latter is present at non-physiological concentrations or is distributed into abnormal cellular compartments.…”