Biology subjects

Hoyerova, K.

Publications and source records attributed to Hoyerova, K..

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↗

bioRxiv2023

Cytokinin Dehydrogenase in Xylem Sap Reveals A Direct Link Between Cytokinin Metabolism and Long-Distance Transport

Metabolic degradation of plant hormones cytokinins (CKs) co-regulates their homeostasis and signalling. In this work, we employed a large-scale bioinformatical analysis to address a diversity of cytokinin oxidase/dehydrogenase (CKX) substrate specificities previously described in several case studies. We present a three-way correlation of the entire CKX amino acid sequences, a variable motif involved in substrate binding, and subcellular localizations predicted by a deep learning model. This correlation is conserved in monocotyledonous plants, suggesting that the CKX diversity in a single species allows a precise tuning of the CK homeostasis. Following these findings, we detected CKX activity in xylem sap for the first time, using the oat (Avena sativa) as a model plant. Further investigation of the substrate specificity and glycosylation of this xylem-located CKX suggested that it originates in roots. We have identified 27 putative CKXs in oats and attributed the xylem-located activity to the extracellular isoforms AsCKX1a,c,d. Finally, we show that the xylem-located CKX activity responds to the nitrate supply, highlighting its physiological relevance. Taken together, we show that CKX directly modulates root-to-shoot CK translocation through metabolic degradation of the transported CKs.

plant biology↗

bioRxiv2020

A role of DAO1 in oxidation of IAA amino acid conjugates revealed through metabolite, high throughput transcript and protein level analysis.

Auxin metabolism is, together with auxin transport, a key determinant of auxin signalling output in plant cells, yet details on the underlying mechanisms and factors involved are still largely unknown. Processes involved in the auxin metabolism are subject to regulation based on numerous signals, including auxin concentration itself. Altered auxin availability and the subsequent changes of auxin metabolite profiles can therefore elucidate the function and regulatory role of individual elements in the auxin metabolic machinery. After analysing auxin metabolism in auxin dependent tobacco BY-2 cell line grown in presence or absence of synthetic auxin 2,4-D we found that both conditions were similarly characterized by very low levels of endogenous indole-3-acetic acid (IAA) and its metabolites. However, metabolic profiling after exogenous application of IAA uncovered that the concentration of N-(2-oxindole-3-acetyl)-L-aspartic acid (oxIAA-Asp), the most abundantly formed auxin metabolite in the control culture, dramatically decreased in auxin-starved conditions. To describe the molecular mechanism behind this regulation, we analysed transcriptome and proteome changes caused by auxin starvation. While no changes in the expression of auxin biosynthetic machinery were observed, many genes related to auxin conjugation and degradation showed differential expression. Selected putative auxin glycosylating enzymes as well as members of the Gretchen Hagen 3 gene family involved in auxin amino acid conjugation showed both up- and down-regulation. Contrarily to that, all tobacco homologs of Arabidopsis thaliana DIOXYGENASE FOR AUXIN OXIDATION 1 (DAO1), known to be responsible for the formation of oxIAA from IAA, showed significant downregulation at both transcript and protein levels. To validate the role of DAO1 in auxin metabolism, we performed auxin metabolite profiling in BY-2 mutants carrying either siRNA-silenced or CRISPR-Cas9-mutated NtDAO1, as well as in dao1-1 Arabidopsis thaliana plants. Both mutants showed not only expectedly lower levels of oxIAA, but also significantly lower abundance of oxidated amino acid conjugates of IAA (oxIAA-Asp). Our results thus represent the first direct evidence on DAO1 activity on IAA amino acid conjugates. Statement of significanceHere we present an analysis of auxin metabolism on metabolite, transcript and protein levels in tobacco BY-2 cell line, collectively identifying oxidation of IAA amino acid conjugates as a new role of DIOXYGENASE FOR AUXIN OXIDATION 1 within an auxin-level-responsive metabolic system.

plant biology↗