Biology subjects

Benkova, E.

Publications and source records attributed to Benkova, E..

13 recordsLinked to original sources

bioRxiv2026

Conserved molecular compatibility for MIZ1 function in cytokinin signaling in land plants

Plant roots adjust their growth direction toward water through hydrotropism, a process that in Arabidopsis thaliana requires the endoplasmic reticulum (ER)-associated protein MIZU-KUSSEI1 (MIZ1). Nonetheless, MIZ1 orthologues occur throughout land plants, including lineages lacking true roots, raising questions about the ancestral function of this protein family. Here, we investigated the evolutionary conservation and functional diversification of MIZ1-like proteins across land plants. MIZ1 orthologues from evolutionarily distant land plants failed to restore hydrotropism or cytokinin sensitivity in the Arabidopsis miz1 mutant, despite conserved protein structure and ER localization. In the liverwort Marchantia polymorpha, loss of MpMIZ1a led to altered cytokinin sensitivity and cytokinin-mediated development in both the sporophyte and gametophyte. These phenotypes and gene expression analyses imply a conserved role of MIZ1 in cytokinin-related processes. Despite this functional similarity, protein-protein docking and bimolecular fluorescence complementation revealed species-specific interactions between MIZ1 proteins and their cognate cytokinin histidine kinase receptors in Arabidopsis and Marchantia. Together, our findings indicate that MIZ1 proteins represent ancient land plant regulators of cytokinin-dependent development whose molecular compatibility and developmental outputs have diverged during evolution. We propose that the role of MIZ1 in root hydrotropism represents a derived recruitment of this ancestral module into root-specific environmental response pathways.

plant biology↗

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↗

bioRxiv2025

Nuclear auxin signalling induces autophagy for developmental reprogramming

The phytohormone auxin is a central regulator of plant growth and development, traditionally known for mediating transcriptional reprogramming through the canonical TIR1/AFB-Aux/IAA signalling pathway. In this study, we reveal that auxin rapidly induces macroautophagy, a catabolic process critical for the removal and recycling of superfluous macromolecules. We demonstrate that natural auxin (IAA) triggers autophagy at physiological concentrations. Genetic and pharmacological analyses show that TIR1/AFB receptors and their adenylate cyclase activity are indispensable for autophagy induction. Furthermore, auxin-induced autophagy depends on transcription, highlighting its integration with the broader gene regulatory networks. Functionally, we show that auxin-induced autophagy is required for efficient developmental reprogramming and organogenesis in both root and shoots. Autophagy is induced at places of local auxin maxima and autophagy-deficient mutants exhibit delayed differentiation and retarded organ primordia progression at the meristematic zones. Together, our findings uncover a dual role for auxin in coordinating gene expression and autophagic clearance, thereby facilitating rapid and effective developmental transitions.

plant biology↗

bioRxiv2024

Super-resolution expansion microscopy in plant roots

Super-resolution methods enable spatial resolution far better than the optical diffraction limit of about half the wavelength of light ([~]200-300 nm) but have yet to attain widespread use in plants, owing in large part to plants challenging optical properties. Expansion microscopy improves effective resolution by isotropically increasing physical distances between sample structures while preserving relative spatial arrangements, and clears the sample. However, its application to plants has been hindered by the rigid, mechanically cohesive structure of plant tissues. Here, we report on whole-mount expansion microscopy of Arabidopsis thaliana root tissues (PlantEx), achieving 4-fold resolution increase over conventional microscopy, highlighting microtubule cytoskeleton organization and interaction between molecularly defined cellular constituents. By combining PlantEx with STED microscopy, we increase nanoscale resolution further and visualize the complex organization of subcellular organelles from intact tissues by example of the densely packed COPI-coated vesicles associated with the Golgi apparatus and put these into cellular structural context.

plant biology↗

bioRxiv2023

Mechanical forces in extendable tissue matrix orient cell divisions via microtubule stabilization in Arabidopsis

Plant morphogenesis relies exclusively on oriented cell expansion and division. Nonetheless, the mechanism(s) determining division plane orientation remain elusive. Here we studied tissue healing after laser-assisted wounding in roots and uncovered how mechanical forces of cell expansion stabilize and reorient microtubule cytoskeleton for orientation of cell division. We revealed that root tissue functions as interconnected cell matrix with a radial gradient of tissue extendibility causing a predictable tissue deformation after wounding. This causes instant redirection of expansion in the surrounding cells and reorientation of microtubule arrays ultimately predicting cell division orientation. Microtubules are destabilized under low tension, whereas stretching of cells, either through wounding or external aspiration immediately induce their polymerisation. The higher microtubule abundance in the stretched cell parts leads to reorientation microtubule arrays and ultimately cell division planes. This provides a long-sought mechanism for flexible re-arrangement of cell divisions by mechanical forces for tissue reconstruction and plant architecture.

cell biology↗

bioRxiv2022

Decoy Receptor Fine-tunes Cytokinin Signaling

Hormone perception and signaling pathways play a fundamental regulatory function in cell growth, developmental, and physiological processes in both plant and animal systems. Those pathways are activated by hormone binding to the receptor to trigger cellular responses. Equally important are mechanisms that suppress activated transduction cascades to reset the system. Different mechanisms at the level of hormone biosynthesis and deactivation through degradation, conjugation, and production of repressors that attenuate transduction cascades downstream of receptors are known. In animal systems, decoy receptors have been identified as another important mechanism for fine-tuning the activity of the signaling pathways in processes like inflammatory responses, apoptosis, and blood vessel formation. Decoy receptors recognize and bind specific signaling molecules, but they cannot activate downstream signaling pathways thus providing competitive inhibition. Here we describe the first decoy receptor in plants. We show that the splicing variant of CRE1/AHK4 receptor of cytokinin, a hormone with a key role in the regulation of cell division and meristem maintenance in plants, acts as a decoy receptor to attenuate cytokinin signaling. We propose that this novel mechanism of signaling control applies in processes when modulation of CK signaling is needed.

plant biology↗

bioRxiv2022

Dual role of Pectin Methyl Esterase activity for plant cell growth regulation.

Acid-growth theory has been postulated in the 70s to explain the rapid elongation of cells in response to plant hormone auxin. More recently, it has been demonstrated that activation of the proton ATPs pump (H+-ATPs) promoting acidification of the apoplast is the principal mechanism through which hormones like auxin as well as brassinosteroids (BR) induce cell elongation. However, the impact of this acidification on the mechanical properties of the cell wall remained largely unexplored. Here, we use Atomic Force Microscopy (AFM) to demonstrate that acidification of apoplast is necessary and sufficient to induce cell elongation through cell wall relaxation. Moreover, we demonstrate that Pectin Methyl Esterase (PME) can induce both cell wall softening or stiffening in extracellular calcium dependent-manner and that tight control of PME activity is required for hypocotyl elongation.

cell biology↗

bioRxiv2021

Design principles of plant root morphogenesis

Plants grow roots to adjust their bodies to dynamic changes in the surrounding environment. How do plant roots emerge from the stem cell reservoir during embryogenesis is however poorly understood. Here, we present a bottom-up strategy to address this challenge by combining empirical observations with advanced computer modeling techniques. We demonstrate that the anisotropy of root growth results from differential growth rates of adjacent tissues, whereas the root meristem development incorporates a multi-level feedback loop between complex transport network of phytohormone auxin, auxin-dependent cell growth and cytoskeleton rearrangements. In silico model predictions are in close agreement with in vivo patterns of anisotropic growth, auxin distribution, and cell polarity, as well as several root phenotypes caused by chemical, mechanical, or genetic perturbations. Our findings reveal a minimal set of design principles connecting tissue mechanics, cell anisotropy, and directional transport that are sufficient for self-organization of the root meristem shape. A mobile auxin signal transported through immobile cells orchestrates polarity and growth mechanics to instruct the morphogenesis of an independent organ.

plant biology↗

bioRxiv2020

Phosphoproteomics after nitrate treatments reveal an important role for PIN2 phosphorylation in control of root system architecture.

Nitrate is an important signaling molecule that commands genome-wide gene expression changes that impact metabolism, physiology, plant growth and development. Although gene expression responses to nitrate at the mRNA level have been characterized in great detail, the impact of nitrate signaling at the proteome level has been much less explored. Most signaling pathways involve post-translational modifications of key protein factors and chiefly among these modifications is protein phosphorylation. In an effort to identify new components involved in nitrate responses in plants, we performed analyses of the Arabidopsis thaliana root phosphoproteome in response to nitrate treatments via liquid chromatography coupled to tandem mass spectrometry. We identified 268 phosphoproteins that show significant changes at 5 min or 20 min after nitrate treatments. The large majority of these proteins (96%) are coded by genes that are not modulated at the expression level in response to nitrate treatments in publicly available transcriptome data. Proteins identified by 5 min include potential signaling-components such as kinases or transcription factors. In contrast, by 20 min, proteins identified were associated with protein binding, transporter activity or hormone metabolism functions. Interestingly, the phosphorylation profile of NITRATE TRANSPORTER 1.1 (NRT1.1) mutant plants in response to nitrate at 5 min was significantly different (95%) as compared to wild-type plants. This result is consistent with the role of NRT1.1 as a key component of a nitrate signaling pathway that involves phosphoproteomic changes. Our integrative bioinformatics analysis highlights auxin transport as an important mechanism modulated by nitrate signaling at the post-translational level. We experimentally validated the role of PIN2 phosphorylation in both primary and lateral root growth responses to nitrate. Our data provide new insights into the phosphoproteome and identifies novel protein components that are regulated post-translationally, such as PIN2, in nitrate responses in Arabidopsis thaliana roots.

plant biology↗

bioRxiv2020

Modulation of root growth by nutrient-defined fine-tuning of polar auxin transport

Nitrogen is an essential macronutrient and its availability in soil plays a critical role in plant growth, development and impacts agricultural productivity. Plants have evolved different strategies to sense and respond to heterogeneous nitrogen distribution. Modulating root system architecture, including primary root growth and branching, is among the most essential plant adaptions to ensure adequate nitrogen acquisition. However, the immediate molecular pathways coordinating the adjustment of root growth in response to varying nitrogen sources are poorly understood. Here, using a combination of physiological, live in vivo high- and super resolution imaging, we describe a novel adaptation strategy of root growth on available nitrogen source. We show that growth, i.e. tissue-specific cell division and elongation rates are fine-tuned by modulating auxin flux within and between tissues. Changes in auxin redistribution are achieved by nitrogen source dependent post-translational modification of PIN2, a major auxin efflux carrier, at an uncharacterized, evolutionary conserved phosphosite. Further, we generate a computer model based on our results which successfully recapitulate our experimental observations and creates new predictions that could broaden our understanding of root growth mechanisms in the dynamic environment.

plant biology↗

bioRxiv2019

Auxin-dependent xyloglucan remodelling defines differential tissue expansion in Arabidopsis thaliana

Size control is a fundamental question in biology, showing incremental complexity in case of plants whose cells possess a rigid cell wall. The phytohormone auxin is a vital growth regulator with central importance for differential growth control. Here we show that growth inducing and repressing auxin conditions correlate with reduced and enhanced complexity of extracellular xyloglucans, respectively. In agreement, genetic interference with xyloglucan complexity distinctly modulates auxin-dependent differential growth rates. Our work proposes that an auxin-dependent, spatially defined effect on xyloglucan structure and its effect on cell wall mechanics specify differential, gravitropic hypocotyl growth.

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

PICKLE recruits RETINOBLASTOMA RELATED 1 to Control Lateral Root Formation in Arabidopsis

Lateral root (LR) formation is an example of plant post-embryonic organogenesis event. LRs are issued from non-dividing cells entering consecutive steps of formative divisions, proliferation and elongation. The chromatin remodeling protein PICKLE negatively regulates auxin-mediated LR formation through a mechanism that is not yet known. Here we show that PICKLE interacts with RETINOBLASTOMA-RELATED 1 (RBR1) to repress the LATERAL ORGAN BOUNDARIES-DOMAIN 16 (LBD16) promoter activity. Since LBD16 function is required for the formative division of LR founder cells, repression mediated by the PKL-RBR1 complex negatively regulates formative division and LR formation. Inhibition of LR formation by PKL-RBR1 is counteracted by auxin indicating that in addition to auxin-mediated transcriptional responses, the fine-tuned process of LR formation is also controlled at the chromatin level in an auxin-signaling dependent manner.

plant biology↗