Biology subjects

Schulze, W. X.

Publications and source records attributed to Schulze, W. X..

11 recordsLinked to original sources

bioRxiv2026

Cryo-EM structure of the Arabidopsis thaliana V-type ATPase

Vacuolar-type ATPases (V-ATPases) are evolutionarily conserved rotary proton pumps that play essential roles in the eukaryotic cell. By coupling ATP hydrolysis in their cytosolic V1 region to proton translocation through their membrane-embedded VO region, V-ATPases establish and maintain an acidic pH in the lumen of several different organelles. Functional diversity in the pump is enabled by multiple paralogous genes for the subunits of the complex, which are expressed in a tissue- and organelle-specific manner. Interactions between V-ATPase and TLDc domain-containing proteins have been shown to regulate the enzyme in yeast and mammals but their relevance in plants has remained unclear. We isolated the endogenous V-ATPase from Arabidopsis thaliana leaves and determined its structure by electron cryomicroscopy. Mass spectrometry showed that most of the enzyme originated from the tonoplast. The structural analysis revealed the full rotary catalytic cycle of the plant V-ATPase, and a combination of structural and biochemical experiments showed S-acylation of subunits AP1 and the tonoplast-specific subunit a3 isoform. A subpopulation of complexes derived from the trans-Golgi network/early endosome was identified and found to bind the TLDc protein OXR5. Together, these findings reveal plant-specific features in V-ATPase and suggest organelle-specific interactions with TLDc proteins, pointing to conserved but context-dependent V-ATPase regulation in eukaryotes.

biochemistry↗

bioRxiv2026

A receptor-like kinase controls plasmodesmal transport of conserved 30K viral movement proteins through phosphorylation

Intercellular viral movement in plants is mediated by movement proteins (MPs) that modulate plasmodesmata (PD) enabling cell-to-cell and systemic trafficking. Although phosphorylation has long been implicated in the regulation of MP localization and activity, the identity of host kinases and the interface with immune signaling have remain unresolved. Here, we identified the Arabidopsis thaliana lectin receptor-like kinase RDA2 as a PD-associated regulatory component of viral movement. Using proximity labeling, we detected RDA2 as a proximal interactor of the tobacco mosaic virus (TMV) MP, and show that RDA2 directly phosphorylates MP at multiple sites in vitro. Phosphorylation at threonine 75 is required for efficient PD targeting and intercellular movement, while phospho-dead mutants failed to complement viral spread. Loss of RDA2 enhanced MP mobility and increased TMV accumulation in planta, indicating that RDA2 modulates PD transport during the infection. RDA2 also interacted with and phosphorylated the movement protein of cucumber mosaic virus, implicating that this regulatory mechanism extends across members of the 30K MP superfamily. Our findings demonstrate that a plasma membrane receptor-like kinase can directly modify viral movement proteins, establishing a mechanistic link between receptor-mediated immune signaling and the post-translational control of symplasmic connectivity. One-sentence summaryRDA2, a lectin receptor-like kinase, directly phosphorylates conserved 30K viral movement proteins to control their plasmodesmal targeting and restrict cell-to-cell viral spread in plants

plant biology↗

bioRxiv2025

Diversity of stomatal and cuticular structures affect microbial colonization in temperate forest tree species

Throughout their life cycle, tree leaves are subject to colonization and degradation by microorganisms, including fungi, bacteria, and algae. These relationships co-evolved with chemical properties, leaf shape, and surface structures. Here we quantified leaf abaxial surface texture features based on variables extracted from scanning electron microscopic images, resulting in an abstract quantitative texture complexity score. This complexity score was used to test functional hypotheses such as growth habitat preferences and microbial colonization patterns. Here we show that the evolution of leaf surface texture complexity traits correlated with anatomical features such as stomatal density and leaf orientation and correlated with Ellenberg temperature habitat indicator. Furthermore, increasing leaf surface texture complexity was found to be negatively correlated with plant pathogen richness (broad-leaved species) or lichenization (conifers), suggesting protection effects. Moreover, we found a negative correlation of leaf surface texture complexity with fungal and bacterial specialists. Our results highlight leaf surface texture complexity as a key, previously underappreciated trait shaping microbial diversity and leaf-microbe interaction patterns. This opens promising avenues for future research on plant-microbe co-evolution, trait-based ecosystem modeling, and the potential use of surface traits in forest management and disease resistance strategies.

plant biology↗

bioRxiv2025

Phosphoproteomics uncovers rapid and specific transition from plant two-component system signaling to Ser/Thr phosphorylation by the intracellular redox sensor AHK5

The Arabidopsis thaliana histidine kinase 5 (AHK5) is proposed to function as a intracellular sensor of active oxygen species (ROS) and redox conditions, fine-tuning responses to diverse ROS-inducing stimuli through a multistep phosphorelay (MSP) system. To elucidate the mechanism of ROS perception by AHK5 and signaling through posttranslational modifications, a comparative phosphoproteomics analysis was conducted using ahk5 loss-of-function and wild type seedlings in response to exogenously applied H2O2. Under control and H2O2-related conditions, a rapid signaling transition from the MSP to Ser/Thr phosphorylation was observed. AHK5 appears to regulate ROS-responsive phosphorylation at the plasma membrane by the modification of nanodomain components such as remorins, aquaporins, IQDs, and patellins, positioning it as a central integrator of redox signaling during adaptation to diverse stresses. AHK5 drives a distinct, ABA-independent signaling pathway for H2O2-induced stomatal closure, likely by regulating phosphorylation of key components such as RBOHD, CAS, and HPCA1. Moreover, AHK5-fine-tuned root development involves ABA-dependent signaling and appears to act through modulation of auxin transport via phosphorylation of auxin carrier, highlighting a mechanistic divergence between ROS responses in the shoot and root. Our data further suggest that AHK5 orchestrates stress-responsive nanodomain-associated signaling hubs through regulation of membrane trafficking, cytoskeletal dynamics, and endocytosis to mediate stress responses. HighlightAHK5 perceives and may amplify H2O2 signaling by linking two-component systems to Ser/Thr phosphorylation, likely by modulating membrane-associated signalosomes, thereby fine-tuning responses to stress-induced reactive oxygen species.

plant biology↗

bioRxiv2025

In situ architecture of plasmodesmata suggests mechanisms controlling intercellular exchange

Plasmodesmata are nanoscopic channels that traverse plant cell walls, establishing membrane and cytoplasmic continuity between adjacent cells to enable direct intercellular exchange. Although numerous components have been identified, their molecular organization and roles in controlling passage remain unclear. Here, we used cryo-electron tomography to resolve the in situ architecture of plasmodesmata in Physcomitrium patens across tissues and physiological states. We show how callose deposition at the cell wall shapes channel architecture to modulate permeability and identify helical protein assemblies formed by Multiple C2 domain and Transmembrane Proteins (MCTPs), which scaffold a central endoplasmic reticulum tubule and tether it to the plasma membrane. These findings define core architectural features of plasmodesmata and establish a structural framework for understanding how membrane, protein and cell wall components coordinate intercellular connectivity in plants.

cell biology↗

bioRxiv2024

Identification of nuclear pore proteins at plasmodesmata

Plasmodesmata (PD) mediate intercellular exchange of small molecules, RNAs and proteins between plant cells with an apparent exclusion limit for passive non-specific transport, and transport of specific cargo mediated by mediators. PD and nuclear pore complexes (NPC) are nanometer sized micropores with strikingly similar properties. Cargo translocation through NPC is mediated by phase separating FG-nucleoporins (FG-NUP). Here, bioinformatics, proteomics and fluorescence imaging identified FG-NUPs at PD. Transient expression of GFP fusions at low and intermediate expression levels supported dual localization of 12 NUPs to NPC and PD. Structured illumination microscopy detected the transmembrane anchor NUP CPR5 close to orifices of PD. cpr5 mutants showed reduced intercellular short-root (SHR) transport. However, transport defects cannot be excluded due to indirect effects in the mutants. Identification of FG-NUPs at PD is consistent with the recruitment of NUPs to form a PD pore gating complex consistent with phase separation domains as diffusion barriers at PD. Further analyses will be required to determine whether NUPs are bona fide PD components, or accumulate at PD in certain conditions, or may serve intermediate NPC storage.

cell biology↗

bioRxiv2023

Nuclear localization of HD-Zip IV transcription factor GLABRA2 is driven byImportin α

GLABRA2 (GL2), a class IV homeodomain leucine-zipper (HD-Zip IV) transcription factor (TF) from Arabidopsis, is a developmental regulator of specialized cell types in the epidermis. GL2 contains a putative monopartite nuclear localization sequence (NLS) partially overlapping with its homeodomain (HD). We demonstrate that NLS deletion or alanine substitution of its basic residues (KRKRKK) affects nuclear localization and results in a loss-of-function phenotype. Fusion of the predicted NLS (GTNKRKRKKYHRH) to the fluorescent protein EYFP is sufficient for its nuclear localization in roots and trichomes. The functional NLS is evolutionarily conserved in a distinct subset of HD-Zip IV members including PROTODERMAL FACTOR2 (PDF2). Despite partial overlap of the NLS with the HD, genetic dissection of the NLS from PDF2 indicates that nuclear localization and DNA binding are separable functions. Affinity purification of GL2 from plant tissues followed by mass spectrometry-based proteomics identified Importin (IMP) isoforms as potential GL2 interactors. NLS structural prediction and molecular docking studies with IMP-3 revealed major interacting residues. Split-ubiquitin cytosolic yeast two-hybrid assays suggest interaction between GL2 and four IMP isoforms from Arabidopsis. Direct interactions were verified in vitro by co-immunoprecipitation with recombinant proteins. IMP triple mutants (imp- 1,2,3) exhibit defects in EYFP:GL2 nuclear localization in trichomes but not in roots, consistent with tissue-specific and redundant functions of IMP isoforms in Arabidopsis. Taken together, our findings provide mechanistic evidence for IMP-dependent nuclear localization of GL2 and other HD-Zip IV TFs in plants. One sentence summaryGLABRA2, a representative HD-Zip IV transcription factor from Arabidopsis, contains an evolutionarily conserved monopartite nuclear localization sequence that is recognized by Importin for translocation to the nucleus, a process that is necessary for cell-type differentiation of the epidermis.

plant biology↗

bioRxiv2023

Imaging of plant calcium-sensor kinase conformation monitors real time calcium decoding in planta

Changes in cytosolic calcium concentration are among the earliest reactions to a multitude of stress cues. Whereas a plethora of calcium-permeable channels may generate distinct calcium signatures and contribute to response specificities, the mechanisms by which calcium signatures are decoded is poorly understood. Here we develop a genetically encoded, FRET-based reporter that visualizes the conformational change of calcium-dependent protein kinases (CDPKs/CPKs), preceding kinase activation, for calcium-dependent AtCPK21 and calcium-independent AtCPK23. In pollen tubes, naturally displaying a physiological calcium range, CPK21-FRET, but not CPK23-FRET, report activity oscillations with similar features to cytosolic calcium, suggesting an isoform-specific calcium dependency and reversibility of the conformational change. In guard cells CPK21-FRET identifies CPK21 as a decoder of signal-specific calcium signatures in response to ABA and flg22. Based on this data, CDPK-FRET stands as a novel approach for tackling real-time live-cell calcium decoding in a multitude of plant developmental and stress responses.

biochemistry↗

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↗

bioRxiv2020

A phospho-switch in the N-terminus of NRT2.1 affects nitrate uptake by controlling the interaction of NRT2.1 with NAR2.1

NRT2.1 can be phosphorylated at five different sites within N- and C-terminus. Here, we provide a systematic functional characterization of phosphorylation at S21 and S28 within the N-terminus of NRT2.1. We used existing phosphoproteomic data sets of nitrate starvation and nitrate resupply to construct a site-specific correlation network identifying kinase candidates to phosphorylate NRT2.1. By this approach, we identified NO_SCPCAPITRATEC_SCPCAP UO_SCPCAPPTAKEC_SCPCAP RO_SCPCAPEGULATORYC_SCPCAP KO_SCPCAPINASEC_SCPCAP 1 (AT5G49770) which itself was regulated by phosphorylation at S839 and S870 within its kinase domain. In the active state, when S839 was dephosphorylated and S870 was phosphorylated, NURK1 was found to interact with NRT2.1 at dephosphorylated S28. Upon that interaction, NURK1 can phosphorylate NRT2.1 at S21. Phosphorylation of NRT2.1 at S21 resulted in low interaction of NRT2.1 with its activator protein NAR2.1. By contrast, phosphorylation of NRT2.1 at S28 by a yet unknown kinase enhanced the interaction with NAR2.1, but inhibited the interaction with NURK1. We propose that serines S21 and S28 are involved in a phospho-switch mechanism and by which the interaction of NRT2.1 with its activator NAR2.1, and thus NRT2.1 activity, is modulated. NURK1 here was identified as the kinase affecting this phospho-switch through phosphorylation of NRT2.1 at S21 leading to inactivation of NRT2.1.

plant biology↗

bioRxiv2019

The systemin signaling cascade as derived from phosphorylation time courses under stimulation by systemin and its inactive Thr17Ala (A17) analog

Systemin is a small peptide with important functions in plant wound response signaling. Although transcriptional responses of systemin action are well described, the precise signaling cascades involved in its perception and signal transduction are poorly understood at the protein level. Here we use a phosphoproteomic profiling study involving stimulation time courses with systemin and its inactive analogon A17 to reconstruct a systemin-specific kinase/phosphatase signaling network. The time course analysis of systemin-induced phosphorylation patterns revealed early events at the plasma membrane, such dephosphorylation of H+-ATPase, rapid phosphorylation of NADPH-oxidase and Ca2+-ATPase. Later responses involved transient phosphorylation of small GTPases and vesicle trafficking proteins, as well as transcription factors. Based on a correlation analysis of systemin-specific phosphorylation profiles, we predict substrate candidates for 56 systemin specific kinases and 18 phosphatases. Among the kinases are several systemin-specific receptor kinases as well as kinases with downstream signaling functions, such as MAP-kinases. A regulatory circuit for plasma membrane H+-ATPase was predicted and confirmed by in-vitro activity assays. In this regulatory model we propose that upon systemin treatment, H+-ATPase LHA1 is rapidly de-phosphorylated at its C-terminal regulatory residue T955 by phosphatase PLL5, resulting in the alkalization of the growth medium within 2 minutes of systemin treatment. We further propose that the H+-ATPase LHA1 is re-activated by MAP-Kinase MPK2 later in the systemin response. MPK2 was identified with increased phosphorylation at its activating TEY-motif at 15 minutes of treatment and the predicted interaction with LHA1 was confirmed by in-vitro kinase assays. Our data set provides a valuable resource of proteomic events involved in the systemin signaling cascade with a focus on predictions of substrates to systemin-specific kinases and phosphatases.

plant biology↗