Biology subjects

Harter, K.

Publications and source records attributed to Harter, K..

17 recordsLinked to original sources

bioRxiv2025

Arabidopsis HYPERSENSITIVE INDUCED REACTION 2 affects plasma membrane receptor pathways and organization

The distribution of proteins across the plasma membrane is not uniform; however, the principles governing their organization remain not fully understood. Hypersensitive-induced reaction (HIR) proteins are plant-specific members of the stomatin/prohibitin/flotillin/HflK/C (SPFH) family that have been shown to influence membrane organization. Arabidopsis thaliana HIR2 interacts with multiple plasma membrane proteins, including receptor kinases such as BAK1-INTERACTING RECEPTORS 2 and 3 (BIR2 and 3), BRI1-ASSOCIATED KINASE 1 (BAK1), FLAGELLIN SENSING 2 (FLS2), and BRASSINOSTEROID INSENSITIVE 1 (BRI1). These interactions connect HIR2 to BAK1-mediated signaling pathways, as evidenced by impaired growth and immunity phenotypes in hir2 mutants. HIR2 is anchored to the inner leaflet of the plasma membrane through a hydrophobic interaction domain and S-acylation. Using single-particle tracking photoactivated localization microscopy (sptPALM), we showed that HIR2 affects receptor kinase dynamics and clustering, suggesting a role in spatially coordinating receptor complex activities. Structural modeling with AlphaFold 3 predicts a multimeric circular cup-like assembly for HIR2, consistent with high molecular weight complexes identified through blue native polyacrylamide gel electrophoresis. These findings indicate that HIR2 forms a discrete membrane compartment, providing a novel structural framework for spatial membrane organization and thereby modulating the function of membrane-resident receptors.

plant biology↗

bioRxiv2024

Contrasting Effects of Cytoskeleton Disruption on Plasma Membrane Receptor Dynamics: Insights from Single-Molecule Analyses

Traditional models such as the fluid mosaic model or the lipid raft hypothesis have shaped our understanding of plasma membrane (PM) organization. However, recent discoveries have extended these paradigms by pointing to the existence of micro- and nanodomains. Here, we investigated the role of the cytoskeleton in general and whether the picket fence model, established in animal cells, is transferable to the plant cell system. By using single-particle tracking photoactivated localization microscopy (sptPALM) in combination with genetically encoded enzymatic tools for the targeted disruption of the cytoskeleton, we studied the dynamics and nanoscale organization of a selection of PM receptor-like kinases (RLKs) and receptor-like proteins (RLPs). Our findings show that the disintegration of actin filaments leads to decreased diffusion, more restrictive motion patterns, and enlarged clusters, whereas the disintegration of microtubules results in increased diffusion, more unconstrained diffusive behavior, and decreased cluster sizes of the tested RLKs and RLPs. These results underscore the potential unique regulatory functions of cytoskeleton components in plants and suggest an altered mechanism compared to the picket fence model of the animal cell system. Our qualitative data can serve as the foundation for further investigations aimed at developing a comprehensive and refined model of protein dynamics and organization in plant cells.

plant biology↗

bioRxiv2024

Simultaneous and Dynamic Super-Resolution Imaging of Two Proteins in Arabidopsis thaliana using dual-color sptPALM

Super-resolution microscopy techniques have revolutionized cell biology by providing insights into single-molecule dynamics and nanoscale organization within living cells. However, the application of dynamic live-cell methods in plants remains limited by the availability of suitable fluorophores for simultaneous visualization of multiple proteins. To address this challenge, we implemented a dual-color single-particle tracking photoactivated localization microscopy (sptPALM) approach based on codon-optimized photoactivatable fluorescent proteins PA-GFP and PATagRFP. Recently, we demonstrated their individual performance in single-color experiments in Nicotiana benthamiana and Arabidopsis thaliana cells. Here, we establish their combined use for dual-color sptPALM, enabling the simultaneous tracking of two distinct protein species within the same plant cell. This approach provides a framework to investigate the coordinated dynamics, interactions, and spatial organization of multiple proteins in living plant cells.

plant biology↗

bioRxiv2023

GreenMemTraX: A User-Friendly Software for Super-Resolution Analysis of Membrane Protein Dynamics and Nanoscale Organization in Plants

Super-resolution microscopy (SRM) approaches revolutionize cell biology by providing insights into the nanoscale organization and dynamics of macromolecular assemblies and single molecules in living cells. A major hurdle limiting SRM democratization is post-acquisition data analysis which is often complex and time-consuming. Here, we present OneFlowTraX, a user-friendly and open-source software dedicated to the analysis of single-molecule localization microscopy (SMLM) approaches such as single-particle tracking photoactivated localization microscopy (sptPALM). Through an intuitive graphical user interface, OneFlowTraX provides an automated all-in-one solution for single-molecule localization, tracking, as well as mobility and clustering analyses. OneFlowTraX allows the extraction of diffusion and clustering parameters of millions of molecules in a few minutes. Finally, OneFlowTraX greatly simplifies data management following the FAIR (Findable, Accessible, Interoperable, Reusable) principles. We provide a detailed step-by-step manual and guidelines to assess the quality of single-molecule analyses. Applying different fluorophores including mEos3.2, PA-GFP, and PATagRFP, we exemplarily used OneFlowTraX to analyze the dynamics of plant plasma membrane-localized proteins including an aquaporin, the brassinosteroid receptor Brassinosteroid Insensitive 1 (BRI1) and the Receptor-Like Protein 44 (RLP44).

plant biology↗

bioRxiv2023

In planta exploitation of leaf apoplastic compounds: a window of opportunity for spatiotemporal studies of apoplastic metabolites, hormones and physiology

Processes in the leaf apoplast are relevant for development, cell wall rheological properties, plant nutrition, sink-source portioning, microbe-host plant-interactions or intercellular information exchange and signaling and are therefore regulated or influenced by the composition of the leaf apoplastic solute. In contrast to the traditional methods for the extraction of apoplastic solutes that are more or less destructive, we propose a new method that allows extraction of leaf apoplastic solutes (i) non-invasively and, thus, (ii) over time. Moreover, the method has (iii) a high spatial resolution that allows identification of solute-microdomains in the leaf apoplast. The method was established for Arabidopsis thaliana and Vicia faba leaves but should also be applicable to other plants species with similar leaf morphologies. It is based on the infiltration of an aqueous extraction solution into the apoplast followed by its recovery seconds later, both through the stomata. By this, the apoplast (and its solutes) of an identical leaf can be sampled on successive days with negligible symplastic contamination. A spatiotemporal mapping of leaf apoplastic ion and metabolite patterns within the identical leaf opens a window of opportunity for understanding apoplast biology. As for example, the existence of apoplastic abscisic acid gradients within a leaf in response to salinity was witnessed in this study, as was the unsuspected accumulation of kaempferol glycosides in the leaf apoplast. The presented method is relevant for plant developmental biologists, phytopathologists, plant physiologists, plant nutritionists and others that need to integrate apoplast biology into their research approaches.

plant biology↗

bioRxiv2023

nf-root: a best-practice pipeline for deep learning-based analysis of apoplastic pH in microscopy images of developmental zones in plantroot tissue

Here we report nextflow-root (nf-root), a novel best-practice pipeline for deep learning-based analysis of fluorescence microscopy images of plant root tissue, aimed at studying hormonal mechanisms associated with cell elongation, given the vital role that plant hormones play in the development and growth of plants. This bioinformatics pipeline performs automatic identification of developmental zones in root tissue images, and analysis of apoplastic pH measurements of tissue zones, which is useful for modeling plant hormone signaling and cell physiological responses. Mathematical models of physiological responses of plant hormones, such as brassinolide, have been successfully established for certain root tissue types, by evaluating apoplastic pH via fluorescence imaging. However, the generation of data for this modeling is time-consuming, as it requires the manual segmentation of tissue zones and evaluation of large amounts of microscopy data. We introduce a high-throughput, highly reproducible Nextflow pipeline based on nf-core standards that automates tissue zone segmentation by implementing a deep-learning module, which deploys deterministically trained (i.e. bit-exact reproducible) convolutional neural network models, and augments the segmentation predictions with measures of prediction uncertainty and model interpretability, aiming to facilitate result interpretation and verification by experienced plant biologists. To train our segmentation prediction models, we created a publicly available dataset composed of confocal microscopy images of A. thaliana root tissue using the pH-sensitive fluorescence indicator, and manually annotated segmentation masks that identify relevant tissue zones. We applied this pipeline to analyze exemplary data, and observed a high statistical similarity between the manually generated results and the output of nf-root. Our results indicate that this approach achieves near human-level performance, and significantly reduces the time required to analyze large volumes of data, from several days to hours.

bioinformatics↗

bioRxiv2023

Cytokinins regulate spatially-specific ethylene production to control root growth in Arabidopsis

The two principal growth regulators cytokinins and ethylene are known to interact in the regulation of plant growth. However, information about underlying molecular mechanism and positional specificity of the cytokinin/ethylene crosstalk in root growth control is scarce. We have identified spatial specificity of cytokinin-regulated root elongation and root apical meristem (RAM) size, both of which we demonstrate to be ethylene biosynthesis-dependent. Upregulation of the cytokinin biosynthetic gene ISOPENTENYLTRANSFERASE (IPT) in proximal and peripheral tissues leads to both root and RAM shortening. In contrast, IPT activation in distal and inner tissues reduces RAM size while leaving the root length comparable to mock-treated controls. We show that cytokinins regulate two steps specific to ethylene biosynthesis, the production of ACC by ACC SYNTHASEs (ACSs), and its conversion to ethylene by ACC OXIDASEs (ACOs). We describe cytokinin- and ethylene-specific regulation controlling the activity of ACSs and ACOs that are spatially discrete along both proximo/distal and radial root axes. Using direct ethylene measurements, we identify ACO2, ACO3 and ACO4 as being responsible for ethylene biosynthesis and the ethylene-regulated root and RAM shortening in cytokinin-treated Arabidopsis. Finally, we describe the tight cooperation between cytokinin and ethylene signaling in cytokinin-induced, ethylene-regulated control of ACO4 due to the direct interaction between ARABIDOPSIS RESPONSE REGULATOR 2 (ARR2), a member of the multistep phosphorelay cascade and the C-terminal portion of ETHYLENE INSENSITIVE 2 (EIN2-C), a key regulator of canonical ethylene signaling.

plant biology↗

bioRxiv2022

A phospho-switch provided by LRR receptor-like kinase, ALK1/QSK1/KIN7, prioritizes ABCG36/PEN3/PDR8 transport toward defense

Based on its proposed substrate preferences, the ABC transporter, ABCG36/PDR8/PEN3, from the model plant Arabidopsis stands at the cross-road between growth and defence. Recently, ABCG36 was shown to export a few indolic compounds, including the auxin precursor, indole-3-butyric acid (IBA), and to be implicated in the export of the major phytoalexin of Arabidopsis, camalexin, although clear-cut proof of camalexin transport activity is still lacking. Here we provide strong evidence that ABCG36 catalyses the direct, ATP-dependent export of camalexin over the plasma membrane, however, most likely in functional interplay with non-camalexin transporting ABCG isoforms. We identify the leucin-rich repeat receptor-like kinase, Auxin-induced LRR Kinase1 (ALK1/KIN7/QSK1), as a functional kinase to physically interact with and phosphorylate ABCG36. ABCG36 phosphorylation by ALK1 represses unilaterally IBA but not camalexin export leading to a prioritization of ABCG transport toward defense. As a consequence, phospho-dead mutants of ABCG36, like alk1 and abcg36 alleles, are hypersensitive toward infection with the root pathogen, F. oxysporum, caused by elevated fungal progression. Our findings indicate a novel, direct regulatory circuit between a receptor kinase and an ABC transporter determining transporter substrate specificity. It appears that growth and defense balance decisions in plants are performed on the transporter level by means of a reversible phospho-switch.

plant biology↗

bioRxiv2022

PEP7 is a ligand for receptor kinase SIRK1 to regulate aquaporins and root growth

Plant receptor kinases constitute a large protein family that regulate various aspects of development and responses to external biotic and abiotic cues. Functional characterization of this protein family and particularly the identification of their ligands remains a major challenge in plant biology. Previously, we identified plasma membrane-intrinsic SUCROSE INDUCED RECEPTOR KINASE 1 (SIRK1) and QIAN SHOU KINASE 1 (QSK1) as a receptor / co-receptor pair involved in regulation of aquaporins in response to osmotic conditions induced by sucrose. Here, we identified a member of the Elicitor Peptide (PEP) family, namely PEP7, as the specific ligand of receptor kinase SIRK1. PEP7 binds to the extracellular domain of SIRK1 with a binding constant of 1.44{+/-}0.79 {micro}M and is secreted to the apoplasm specifically in response to sucrose treatment. Stabilization of a signaling complex involving SIRK1, QSK1 and aquaporins as substrates is mediated by alterations in the external sucrose concentration or by PEP7 application. Moreover, the presence of PEP7 induces the phosphorylation of aquaporins in vivo and enhance water influx into protoplasts. The loss-of-function mutant of SIRK1 is not responsive to external PEP7 treatment regarding kinase activity, aquaporin phosphorylation and water influx activity. Our data indicate that the PEP7/SIRK1/QSK1 complex represents a crucial perception and response module mediating sucrose-controlled water flux in plants.

plant biology↗

bioRxiv2021

AHK5 mediates ETR1-initiated multistep phosphorelay in Arabidopsis

Plants, like other sessile organisms, need to sense many different signals, and in response to them, modify their developmental programs to be able to survive in a highly changing environment. The multistep phosphorelay (MSP) in plants is a good candidate for a response mechanism that integrates multiple signal types both environmental and intrinsic in origin. Recently, ethylene was shown to control MSP activity via the histidine kinase (HK) activity of ETHYLENE RESPONSE 1 (ETR1)1,2, but the underlying molecular mechanism still remains unclear. Here we show that although ETR1 is an active HK, its receiver domain (ETR1RD) is structurally and functionally unable to accept the phosphate from the phosphorylated His in the ETR1 HK domain (ETR1HK) to initiate the phosphorelay to ARABIDOPSIS HISTIDINE-CONTAINING PHOSPHOTRANSMITTERs (AHPs), the next link downstream members in MSP signaling. Instead, ETR1 interacts with another HK ARABIDOPSIS HISTIDINE KINASE 5 (AHK5) and transfers the phosphate from ETR1HK through the receiver domain of AHK5 (AHK5RD), and subsequently to AHP1, AHP2 and AHP3, independently of the HK activity of AHK5. We show that AHK5 is necessary for ethylene-initiated, but not cytokinin-initiated, MSP signaling in planta and that it thus mediates hormonal control of root growth.

plant biology↗

bioRxiv2020

Arabidopsis AZG2, an auxin induced putative cytokinin transporter, regulates lateral root emergence

The phytohormones cytokinin (CK) and auxin are key regulators of plant growth and development. During the last decade specialised transport mechanisms turned out to be the key for the control of local and long distance hormone distributions. In contrast to auxin, CK transport is poorly understood. Here we show that Arabidopsis thaliana AZG2, a member of the AZG purine transporter family, acts as CK transporter involved in the determination of the root system architecture. The expression of AtAZG2 is primarily auxin dependent and restricted to a small group of cells surrounding the lateral root primordia. Compared to wild type, mutants carrying loss-of-function alleles of Atazg2 have higher density of lateral roots, suggesting AZG2 as being part of a regulatory pathway in lateral root emergence. Moreover, azg2 mutants are partially insensitive to exogenously applied CK, which is consistent with the observation that the CK reporter gene TCSnpro:GFP showed lower fluorescence signal in the roots of azg2 mutants compared to those of wild type. These results indicate a defective CK signalling pathway in the region of lateral root primordia. By the integration of AtAZG2 subcellular localization and CK transport capacity data, our results allowed us to propose a local Auxin/CK signalling model for the regulation of lateral root emergence.

plant biology↗

bioRxiv2019

Efficient data management infrastructure for the integration of imaging and omics data in life science research

As technical developments in omics and biomedical imaging drive the increase in quality, modality, and throughput of data generation in life sciences, the need for information systems capable of integrative, long-term storage and management of these heterogeneous digital assets is also increasing. Here, we propose an approach based on principles of Service Oriented Architecture design, to allow the integrated management and analysis of multi-omics and biomedical imaging data. The proposed architecture introduces an interoperable image management system, the OMERO server, into the backend of qPortal, a FAIR-compliant web-based platform for omics data management. The implementation of an integrated metadata model, the development of software components to enable the communication with the OMERO server, and an extension to the data management operations of established software, allows for FAIR management of heterogeneous omics and biomedical imaging data within an integrated system, which facilitates metadata queries from web-based scientific applications. The applicability of the proposed architecture is demonstrated in two prototypical use cases, a plant biology study using confocal scanning microscopy, and a clinical study on hepatocellular carcinoma, with data from a variety of medical imaging and omics modalities. We anticipate that FAIR data management systems for multi-modal data repositories will play a pivotal role in data-driven research, as the joint analysis of omics and imaging data becomes not only desirable but necessary to derive novel insights into biological processes. In particular for powerful machine learning applications where the availability of large datasets with high quality phenotypic annotations is a requirement.

bioinformatics↗

bioRxiv2019

OBERON3 and SUPPRESSOR OF MAX2 1-LIKE proteins form a regulatory module specifying phloem identity

Spatial specificity of cell fate decisions is central for organismal development. The phloem tissue mediates long-distance transport of energy metabolites along plant bodies and is characterized by an exceptional degree of cellular specialization. How a phloem-specific developmental program is implemented is, however, unknown. Here we reveal that the ubiquitously expressed PHD-finger protein OBE3 forms a central module with the phloem- specific SMXL5 protein for establishing phloem identity in Arabidopsis thaliana. By protein interaction studies and phloem-specific ATAC-seq analyses, we show that OBE3 and SMXL5 proteins form a complex in nuclei of phloem stem cells where they establish a phloem-specific chromatin profile. This profile allows expression of OPS, BRX, BAM3, and CVP2 genes acting as mediators of phloem differentiation. Our findings demonstrate that OBE3/SMXL5 protein complexes establish nuclear features essential for determining phloem cell fate and highlight how a combination of ubiquitous and local regulators generate specificity of developmental decisions in plants.

plant biology↗

bioRxiv2019

Three-fluorophore FRET-FLIM enables the study of trimeric protein interactions and complex formation with nanoscale resolution in living plant cells

Protein-protein interaction studies provide valuable insights into cellular signaling. The well-studied brassinosteroid (BR) signaling is initiated by the hormone-binding receptor Brassinosteroid Insensitive 1 (BRI1) and its co-receptor BRI1 Associated Kinase 1 (BAK1). BRI1 and BAK1 were shown to interact independently with the Receptor-Like Protein 44 (RLP44), which is implicated in BRI1/BAK1-dependent cell wall integrity perception. To demonstrate the proposed complex formation of BRI1, BAK1 and RLP44, we established three-fluorophore intensity-based spectral Forster resonance energy transfer (FRET) and FRET-fluorescence lifetime imaging microscopy (FLIM) for living plant cells. Our evidence indicates that RLP44, BRI1 and BAK1 form a ternary complex in a distinct plasma membrane nanodomain. In contrast, although the immune receptor Flagellin Sensing 2 (FLS2) also forms a heteromer with BAK1, the FLS2/BAK1 complexes are localized in other nanodomains. In general, our three-fluorophore FRET approaches provide a feasible basis for studying the interaction and sub-compartmentalization of proteins in great detail.

plant biology↗

bioRxiv2019

A novel mutant allele uncouples brassinosteroid-dependent and independent functions of BRI1

Plants depend on an array of cell surface receptors to integrate extracellular signals with developmental programs. One of the best-studied receptors is BRASSINOSTEROID INSENSITIVE 1 (BRI1), which upon binding of its hormone ligands forms a complex with shape-complimentary co-receptors and initiates a signal transduction cascade leading to a wide range of responses. BR biosynthetic and receptor mutants have similar growth defects on the macroscopic level, which had initially led to the assumption of a largely linear signalling pathway. However, recent evidence suggests that BR signalling is interconnected with a number of other pathways through a variety of different mechanisms. We recently described that feedback information from the cell wall is integrated at the level of the receptor complex through interaction with RLP44. Moreover, BRI1 is required for a second function of RLP44, the control of procambial cell fate. Here, we report on a BRI1 mutant, bri1cnu4, which differentially affects canonical BR signalling and RLP44 function in the vasculature. While BR signalling is only mildly impaired, bri1cnu4 mutants show ectopic xylem in the position of procambium. Mechanistically, this is explained by an increased association of RLP44 and the mutated BRI1 protein, which prevents the former from acting in vascular cell fate maintenance. Consistent with this, the mild BR response phenotype of bri1cnu4 is a recessive trait, whereas the RLP44-mediated xylem phenotype is semi-dominant. Our results highlight the complexity of plant plasma membrane receptor function and provide a tool to dissect BR signalling-related roles of BRI1 from its non-canonical functions.\n\nOne sentence summaryA novel mutant allows to dissect brassinosteroid signalling related and non-canonical functions of the receptor-like kinase BRI1.

plant biology↗

bioRxiv2019

The striking flower-in-flower phenotype of Arabidopsis thaliana Nossen (No-0) is caused by a novel LEAFY allele

SummaryThe transition to reproduction is a crucial step in the life cycle of any organism. In Arabidopsis thaliana the establishment of reproductive growth can be divided into two phases: In the first phase, cauline leaves with axillary meristems are formed and internode elongation begins. In the second phase, lateral meristems develop into flowers with defined organs. Floral shoots are usually determinate and suppress the development of lateral shoots. Here, we describe a Ds transposon insertion mutant in the Nossen (No-0) accession with severe defects in floral development and flower morphology. The most striking aspect is the outgrowth of stems from the axillary bracts of the primary flower carrying terminal secondary flowers. Therefore, we named this mutant flower-in-flower (fif). However, the insertion of the transposon in the annotated gene is not responsible for the fif phenotype. By means of classical and genome sequencing-based mapping, the mutation responsible for the fif phenotype was found to be in the LEAFY (LFY) gene. The mutation, a G-to-A exchange in the second exon of LFY, creates a novel lfy allele and causes a cysteine-to-tyrosine exchange in the 1-helix of the LFY DNA-binding domain. Whereas subcellular localization and homomerization are not affected, the DNA-binding of LFYFIF is abolished. We propose that the amino acid exchange interferes with the cooperative binding of LFY to its target DNA. To generate the strong fif phenotype, LFYFIF may act dominant-negatively by either forming non-binding LFY/LFYFIF heteromers or by titrating out the interaction partners, required for LFY function as transcription factor. Significant StatementThe fif phenotype of Arabidopsis thaliana No-0 is caused by a novel allele of the LEAFY gene

plant biology↗

bioRxiv2018

Integration of Brassinosteroid and Phytosulfokine Signalling Controls Vascular Cell Fate in the Arabidopsis Root

Multicellularity arose independently in plants and animals, but invariably requires robust determination and maintenance of cell fate. This is exemplified by the highly specialized water-and nutrient-conducting cells of the plant vasculature, which are specified long before their commitment to terminal differentiation. Here, we show that the hormone receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) is required for root vascular cell fate maintenance, as BRI1 mutants show ectopic xylem in procambial position. However, this phenotype is unrelated to classical brassinosteroid signalling outputs. Instead, BRI1 is required for the expression and function of its interaction partner RECEPTOR-LIKE PROTEIN 44 (RLP44), which, in turn, associates with the receptor for the peptide hormone phytosulfokine (PSK). We show that PSK signalling is required for the maintenance of procambial cell identity and is quantitatively controlled by RLP44, which promotes complex formation between the receptor for PSK and its co-receptor. Mimicking the loss of RLP44, PSK-related mutants show ectopic xylem in the position of procambium, whereas rlp44 can be rescued by exogenous PSK. Based on these findings, we propose that RLP44 controls cell fate by connecting BRI1 and PSK signalling, providing a mechanistic framework for the integration of signalling mediated by the plethora of plant receptor-like kinases at the plasma membrane.

plant biology↗