Biology subjects

Plihal, O.

Publications and source records attributed to Plihal, O..

4 recordsLinked to original sources

bioRxiv2026

A tonoplast cytokinin riboside transporter gates intracellular hormone availability at the plant-microbe interface

Cytokinin ribosides are major mobile and precursor forms of cytokinins, plant hormones whose transport and subcellular distribution shape developmental and stress responses. Here, we identify Arabidopsis thaliana EQUILIBRATIVE NUCLEOSIDE TRANSPORTER1 (ENT1) as a tonoplast-localized cytokinin riboside transporter. Tissue-specific subcellular analysis under native regulatory elements localized ENT1 predominantly to the tonoplast of root epidermal and lateral root cap cells, where it gates intracellular cytokinin riboside availability. Accordingly, ENT1 overexpression enhanced cytokinin riboside sensitivity and signalling, whereas loss of ENT1 altered adenosine metabolism and disrupted cytokinin homeostasis, leading to the accumulation of multiple zeatin-type cytokinins. ENT1-dependent cytokinin riboside compartmentalization was required for beneficial microbe-induced protection, as ent1 mutants failed to acquire protection against the fungal pathogen Botrytis cinerea and the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. These findings reveal a vacuolar gatekeeping mechanism that controls intracellular cytokinin riboside availability and links hormone compartmentalization to beneficial microbe-dependent plant defence.

plant biology↗

bioRxiv2024

Comprehensive Model of Cell-to-Cell Cytokinin Transport Reveals A Specific Mode of Cytokinin Riboside Influx

1Ribosylated forms of plant hormones cytokinins (CKs) are the dominant CK species translocated at long distances. Their particular roles in plant physiology imply the existence of a yet uncharacterized CK riboside-specific membrane transport system. In this work, we report significant differences in the kinetics of the membrane transport of CK nucleobases and ribosides and the overall affinity of membrane-bound carriers towards the two CK forms. We show that CK ribosides can inhibit the uptake of CK nucleobases in tobacco Bright Yellow 2 cell suspensions but not vice versa, confirming the existence of a membrane transport system that strictly recognizes CK ribosides. We further characterize the membrane transport of CK nucleobases and ribosides mediated by AtENT3 (EQULIBRATIVE NUCLEOSIDE TRANSPORTER 3), showing its preference towards trans-zeatin riboside (tZR) over isopentenyl adenosine (iPR). With the molecular docking and molecular dynamics, we assess the interactions among the side chain of tZR and AtENT3 residues Tyr61 and Asp129, which are conserved in all AtENTs but not in the ENTs from non-plant species. Lastly, we show that atent3 mutation affects shoot phenotype, demonstrating the impact of CK riboside membrane transport on shoot development.

plant biology↗

bioRxiv2019

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

The plant hormone cytokinin regulates various cell and developmental processes, including cell division and differentiation, embryogenesis, activity of shoot and root apical meristems, formation of shoot and root lateral organs and others 1. Cytokinins are perceived by a subfamily of sensor histidine kinases (HKs), which via a two-component phosphorelay cascade activate transcriptional responses in the nucleus. Based on the subcellular localization of cytokinin receptors in various transient expression systems, such as tobacco leaf epidermal cells, and membrane fractionation experiments of Arabidopsis and maize, the endoplasmic reticulum (ER) membrane has been proposed as a principal hormone perception site 2-4. Intriguingly, recent study of the cytokinin transporter PUP14 has pointed out that the plasma membrane (PM)-mediated signalling might play an important role in establishment of cytokinin response gradients in various plant organs 5. However, localization of cytokinin HK receptors to the PM, although initially suggested 6, remains ambiguous. Here, by monitoring subcellular localizations of the fluorescently labelled natural cytokinin probe iP-NBD 7 and the cytokinin receptor ARABIDOPSIS HISTIDINE KINASE 4 (CRE1/AHK4) fused to GFP reporter, we show that pools of the ER-located cytokinin fluoroprobes and receptors can enter the secretory pathway and reach the PM. We demonstrate that in cells of the root apical meristem, CRE1/AHK4 localizes to the PM and the cell plate of dividing meristematic cells. Brefeldin A (BFA) experiments revealed vesicular recycling of the receptor and its accumulation in BFA compartments. Our results provide a new perspective on cytokinin signalling and the possibility of multiple sites of perception at PM and ER, which may determine specific outputs of cytokinin signalling.

plant biology↗

bioRxiv2019

Cell-surface receptors enable perception of extracellular cytokinins

Cytokinins are mobile multifunctional plant hormones with roles in development and stress resilience 1,2. Although cytokinin receptors are substantially localised to the endoplasmic reticulum 3-5, the cellular sites of cytokinin perception continue to be debated 1,6,7. Several cytokinin types display bioactivity 8,9 and recently a cell-specific cytokinin gradient was reported in roots 10. Yet, the importance of spatially heterogeneous cytokinin distribution and the specific cytokinin(s) that account for the different responses remain unclear. Here we show that cytokinin perception by plasma membrane receptors is an effective path for cytokinin response in root cells. Readout from a Two Component Signalling cytokinin-specific reporter (TCSn::GFP;11) is closely matched to intracellular cytokinin content, yet a proportion of bioactive cytokinins are detected in the extracellular fluid. Using cytokinins covalently linked to beads that could not pass the plasma membrane, we demonstrate that strong TCSn activation still occurs and that this response is greatly diminished in cytokinin receptor mutants. Although intracellular receptors play significant roles, we argue for a revision of concepts of cytokinin perception to include the spatial dimensions. In particular, selective ligand-receptor affinities, cellular localisation and tissue distribution of bioactive cytokinins, their receptors, transporters and inactivation enzymes appear all to be components of the signalling regulatory mechanisms.

plant biology↗