Cytokinin fluoroprobe and receptor CRE1/AHK4 localize to both plasma membrane and endoplasmic reticulum

Abstract

30 31 32 33 The plant hormone cytokinin regulates various cell and developmental processes, 34 including cell division and differentiation, embryogenesis, activity of shoot and root 35 apical meristems, formation of shoot and root lateral organs and others 1 . Cytokinins 36 are perceived by a subfamily of sensor histidine kinases (HKs), which via a two-37 component phosphorelay cascade activate transcriptional responses in the nucleus. 38 Based on the subcellular localization of cytokinin receptors in various transient 39 expression systems, such as tobacco leaf epidermal cells, and membrane fractionation 40 experiments of Arabidopsis and maize, the endoplasmic reticulum (ER) membrane has 41 been proposed as a principal hormone perception site 2-4 . Intriguingly, recent study of 42 the cytokinin transporter PUP14 has pointed out that the plasma membrane (PM)-43 mediated signalling might play an important role in establishment of cytokinin response 44 gradients in various plant organs 5 . However, localization of cytokinin HK receptors to 45 the PM, although initially suggested 6 , remains ambiguous. Here, by monitoring 46 subcellular localizations of the fluorescently labelled natural cytokinin probe iP-NBD 7 47 and the cytokinin receptor ARABIDOPSIS HISTIDINE KINASE 4 (CRE1/AHK4) 48 fused to GFP reporter, we show that pools of the ER-located cytokinin fluoroprobes and 49 receptors can enter the secretory pathway and reach the PM. We demonstrate that in 50 cells of the root apical meristem, CRE1/AHK4 localizes to the PM and the cell plate of 51 dividing meristematic cells. Brefeldin A (BFA) experiments revealed vesicular recycling 52 of the receptor and its accumulation in BFA compartments. Our results provide a new 53 perspective on cytokinin signalling and the possibility of multiple sites of perception at 54 PM and ER, which may determine specific outputs of cytokinin signalling. 55 56Fluorescently labelled analogues of phytohormones including auxin, gibberellin, 57 brassinosteroid and strigolactone have been successfully used to map the intracellular fate of 58 their receptors in planta 8 . To adopt this tool for mapping subcellular localization of cytokinin 59 receptors, using docking experiments and cytokinin activity screening bioassays, we selected 60 a fluorescently labelled bioactive compound that interacts with the binding site of a cytokinin 61 receptor. To minimize the possibility of metabolic conversion by cytokinin deactivation 62 enzymes in planta, isopentenyladenine (iP) was used as a natural cytokinin that cannot be 63 transformed through O-glycosylation at the cytokinin side chain. Furthermore, attachment of 64 7-nitro-2,1,3-benzoxadiazole (NBD), a small fluorophore, to the N9 position of iP eliminates 65 a risk of a metabolic conversion of the final cytokinin fluorescent probe iP-NBD ( Fig. 1a) 66 through N-glycosylation, or formation of cytokinin nucleotides. Docking simulations using 67 the CRE1/AHK4-iP crystal structure 9 suggested that iP-NBD may be fully embedded into the 68 active sites ...

Cytokinin isopentenyladenine and its glucoside isopentenyladenine‐9G delay leaf senescence through activation of cytokinin‐associated genes

“…CKNGs have been previously shown to be located largely in the apoplast and vacuole in leaves (Jiskrová et al, 2016), while CK receptors are primarily localized to the endoplasmic reticulum (Caesar et al, 2011;Wulfetange et al, 2011). Recent work, though, suggests CK receptors also appear to be present at the plasma membrane under certain conditions (Kubiasová et al, 2020), possibly placing them in a better position to interact with CKNGs.…”