Biology subjects

Roosjen, M.

Publications and source records attributed to Roosjen, M..

12 recordsLinked to original sources

bioRxiv2026

Phytosulfokine signaling modulates salt stress responses and cell wall remodeling in Arabidopsis

Salinity severely impairs plant growth and development. Increasing evidence suggests that the cell wall (CW) plays a central role in salt stress acclimation, not only as a structural barrier but also as a dynamic sensor that activates downstream stress signaling pathways. To identify extracellular factors involved in cell wall remodeling and integrity signaling during salt stress, we performed a comparative proteomic analysis of the Arabidopsis seedling apoplast. Among the proteins that accumulated under salt stress, we focused on three members of the Subtilisin-Like Protease family (SBT1.1, SBT1.6, and SBT5.3), which have previously been implicated in the processing of signaling peptides and CW-associated proteins. Functional analyses revealed that sbt1.1 and sbt5.3 mutants exhibit enhanced lignin deposition under salt stress, suggesting altered CW remodeling during stress application. Given the established role of SBT1.1 in processing PHYTOSULFOKINE (PSK) peptides, we investigated the involvement of PSK signaling in salt stress responses. Mutants lacking the two PSK RECEPTORS (pskr1 pskr2) displayed reduced growth and increased lignification under salt treatment. Exogenous application of PSK attenuated salt-induced responses, including MITOGEN ACTIVATED PROTEIN KINASE 6 (MPK6) phosphorylation, salt stress marker gene expression, and lignin accumulation, ultimately promoting root elongation under salt stress. Furthermore, PSK treatment mitigated salt-induced changes in CW composition. In cell wall integrity (CWI) mutants, PSK treatment failed to restore wild-type root growth under salt stress and induced a pronounced root bending phenotype in fer-4, indicating that CWI signaling influences PSK-mediated root growth responses. Together, these results support a role for PSK signaling in modulating CW-associated responses to salinity stress in Arabidopsis.

plant biology↗

bioRxiv2026

Systematic proteomics identifies a conserved mechanism for polar targeting of plant cortical proteins

Multicellular development is tightly coupled to the polarization of individual cells, which partitions polar proteins along the cell cortex and can control asymmetric cell division, anisotropic growth, local differentiation or physiology. Mechanisms driving cell polarization have been described in fungi and animals, but these lack counterparts in plants. While several polarized proteins have been identified in plants, the overall mechanisms guiding their polar localization are poorly characterized. Through iterative affinity proteomics on the recently identified SOSEKI polar proteins, we discovered a network of polar proteins that is conserved in the flowering plant Arabidopsis and the liverwort Marchantia. We next used this collection of novel polarized proteins for systematic proximity ligation proteomics in these two species to map their global polar proteome. We identified a subfamily of polarized Protein S-acyl transferase (PAT) enzymes that are required for membrane targeting of Arabidopsis SOSEKI proteins. Using human and fruit fly models, we showed that PAT19 is sufficient for membrane targeting of SOSEKI1, likely through direct palmitoylation. This work demonstrates a conserved mechanism for polar protein targeting in plants and offers a resource for studying polar protein localization.

plant biology↗

bioRxiv2026

Structural and functional insights into the role of Cysteine-Rich Receptor-Like Kinase 18 (CRK18) in Arabidopsis

Plants perceive and integrate diverse environmental signals through receptor kinase (RK) networks at the plasma membrane. Within this, Cysteine-Rich Receptor-Like Kinases (CRKs) constitute a large but not well-understood subfamily characterised by extracellular domains (ECDs) enriched in cysteine residues. CRKs have been implicated in plant responses to biotic and abiotic stress, as well as in developmental processes. Additionally, several CRKs have been proposed to act as redox sensors. Here, we investigate the homodimerization mechanism of Arabidopsis CRK18 and its regulation by redox conditions. By modulating pH and redox state, we assessed the stability and binding dynamics of the CRK18 ECD and its cysteine mutants. We also tested the plasma membrane localisation of all the cysteine mutants involved in the predicted disulfide bonds, and only CRK18C227A, C228A-ECD resembled the plasma membrane localization of wild-type CRK18. We combine co-immunoprecipitation, Forster resonance energy transfer-fluorescence lifetime imaging microscopy and microscale thermophoresis to quantify CRK18 self-association in planta and in vitro. Furthermore, we place CRK18 dimerization in a broader signalling context by identifying CRK18 interaction partners and CRK18-dependent signalling outputs. Using Arabidopsis CRK18 overexpression lines, we perform (phospho)proteomic and immunoprecipitation-mass spectrometry (IP-MS) analyses to map CRK18-centred signalling networks associated with stress-related responses. The constitutive activation of the CRK18 kinase domain and its interaction with many putative (cell wall) glycan-sensing RKs, including PERK15, suggest a regulatory role for CRK18. The absence of significant changes in the proteome and phosphoproteome of CRK18 overexpression lines in the absence of any trigger, and its restricted mobility after elicitation, suggest that CRK18 requires a stimulus for activation and possibly induces membrane microdomain reorganisation. This is in line with the infection assay with the nematode Heterodera schachtii, which causes modification and targeted damage to plant cell walls during infection, revealing that CRK18 acts as negative regulator of this process.

biochemistry↗

bioRxiv2026

Subnuclear cofactor partitioning underlies auxin-dependent transcriptional regulation

Cellular and organismal function relies on the precise activation and repression of gene expression by DNA-binding transcription factors (TFs). Many TFs occur in large gene families, and a key question in biology is how divergent functions emerge in TF families during evolution. Here we discover the biochemical mechanism for transcriptional activation by the Marchantia polymorpha AUXIN RESPONSE FACTOR1 (MpARF1) TF, which relies on direct recruitment of the Mediator complex into subnuclear MpARF1 clusters. We find that the Mediator recruitment region was the evolutionary innovation that converted ARF repressors into activators, switching binding specificity from co-repressor to co-activator. We demonstrate that this evolutionary innovation can be recreated, thereby revealing a deeply conserved mechanism based on competition between ARF clusters at the heart of the transcriptional auxin response.

plant biology↗

bioRxiv2026

Redox-dependent extracellular interaction networks of Cysteine-Rich Receptor-Like Kinases

Reactive oxygen species (ROS) regulate plant development and immunity, but how extracellular ROS signals are decoded and whether the Cysteine-rich Receptor-like Kinases (CRKs) truly serve as the long-suspected ROS sensors remains uncertain. Here, we combine high-throughput interactomics, redox proteomics, structural modelling, and genetics to map a ROS-dependent CRK interaction landscape in Arabidopsis thaliana. Using a redox-dependent interactome assay (RIACRK) on 40 CRK extracellular domains (ECDs), we identified ROS-modulated dimerisation networks with enhanced inter-community connectivity and hub redistribution in the presence of ROS. Integrating this with developmental and flg22-induced expression profiles reveals spatiotemporally limited subnetworks that likely function during activated immunity and leaf senescence, both of which are associated with extensive ROS production. Differential cysteine alkylation coupled with mass spectrometry shows that cysteines in a subset of CRK ectodomains undergo ROS-dependent oxidation. Notably, solvent-exposed, vicinal cysteines C228/C229 in CRK28 emerge as prime redox-sensitive candidates. CRK28 homodimerises and heterodimerises with CRK17 in vivo, and mutation of C228/C229 retains plasma membrane localisation but abolishes CRK28 homodimerisation, indicating a redox-controlled dimerisation switch. Loss of CRK28 delays senescence, while CRK28 overaccumulation from its native promoter causes dwarfism, premature senescence, autoimmune-like phenotypes, extensive phosphoproteome rewiring, and associations with Pathogenesis Related (PR) proteins, ROS-detoxifying enzymes, receptor(-like) kinases, and vesicle trafficking components. These results indicate that CRK28 is a potential ROS-regulated hub connecting extracellular redox signals to CRK network organisation, immune response, and age-related senescence.

plant biology↗

bioRxiv2026

High resolution, proteome-wide mapping of subcellular protein localization in plants

Protein function is intimately connected to subcellular localization, and experimental determination of protein localization is a key element of understanding biological roles. However, even in the best-studied model plants, such as Arabidopsis thaliana, a minority of proteins has an experimentally defined subcellular localization. We present an experimental strategy to globally map plant subcellular proteomes by mass spectrometry. We annotated subcellular localization of 7815 proteins in Arabidopsis roots, 4672 in Arabidopsis seedlings, and 2782 in the liverwort Marchantia polymorpha. By independent validation, we find that these annotations are highly predictive and can be integrated with other proteomics datasets. Cross-species comparisons reveal substantial global conservation of subcellular localization. Furthermore, we demonstrate that the same approach can be used to identify dynamically translocating proteins upon treatment or in a mutant. This work shows the power of global spatial proteome mapping in plants and offers an extensive resource for protein subcellular localization in plants. HighlightsO_LIOptimized approach for global mapping of protein subcellular localization by differential centrifugation in plants C_LIO_LIInteractive resource of subcellular localization of plant proteins at unprecedented depth and resolution C_LIO_LICross-species comparison reveals that the plant subcellular proteome is deeply conserved C_LIO_LIComparative subcellular proteomics of a Brefeldin A treatment and a gnom mutant robustly describes global shifts in protein localization C_LI

plant biology↗

bioRxiv2022

An ultra-fast, proteome-wide response to the plant hormone auxin

The plant signaling molecule auxin controls growth and development through a simple nuclear pathway that regulates gene expresion. There are however several cellular and physiological responses to auxin that occur within seconds, far too rapid to be mediated by transcriptional changes, for which no molecular mechanism has yet been identified. Using a phosphoproteomic strategy in Arabidopsis thaliana roots, we identify an ultra-rapid auxin response system that targets over 1700 proteins, many within 30 seconds. Auxin response is chemically specific, requires known auxin-binding proteins, and targets various pathways. Through exploring its temporal dynamics, we infer auxin-triggered kinase-substrate networks and identify apoplastic pH changes as a target of signaling and as part of a relay mechanism. By generating a variety of phosphoproteomic datasets, integrated with structural information in a web-app, and by demonstrating analysis and inference strategies, we offer a resource to explore rapid and dynamic signaling in plants.

plant biology↗

bioRxiv2022

A RAF-like kinase mediates a deeply conserved, ultra-rapid auxin response

The plant signaling molecule auxin triggers both fast and slow cellular responses across the plant kingdom, including both land plants and algae. A nuclear response pathway mediates auxin-dependent gene expression, and controls a range of growth and developmental processes in land plants. It is unknown what mechanisms underlie both the physiological responses occurring within seconds, and the responses in algae, that lack the nuclear auxin response pathway. We discovered an ultra-fast proteome-wide phosphorylation response to auxin across 5 land plant and algal species, converging on a core group of shared target proteins. We find conserved rapid physiological responses to auxin in the same species and identified a RAF-like protein kinase as a central mediator of auxin-triggered phosphorylation across species. Genetic analysis allowed to connect this kinase to both auxin-triggered protein phosphorylation and a rapid cellular response, thus identifying an ancient mechanism for fast auxin responses in the green lineage.

plant biology↗

bioRxiv2022

Cooperative action of separate interaction domains promotes high-affinity DNA binding of Arabidopsis thaliana ARF transcription factors

The signaling molecule auxin is pivotal in coordinating many growth and development processes in plants mainly through the modulation of gene expression. The transcriptional response to auxin is mediated by the family of auxin response factors (ARF). Monomers of this family recognize a DNA motif (TGTC[TC]/[GG]) called the auxin-response element (AuxRE). ARFs can homodimerize through their DNA binding domains (DBD) thereby enabling cooperative binding for a bipartite inverted AuxRE (IR7). In addition to the DBD, most ARFs contain a C-terminal Phox and Bem1p (PB1) domain both capable of homotypic interactions, and mediating interactions with Aux/IAA repressors. Given the dual role of the PB1 domain, and the ability of both DBD and PB1 domain to mediate dimerization, a key question is how each of these domains contributes to conferring DNA-binding specificity and affinity. So far, ARF-ARF and ARF-DNA interactions have mostly been approached using qualitative methods that do not provide a quantitative and dynamic view on the binding equilibria. Here, we utilize a DNA binding assay based on single-molecule Forster resonance energy transfer (smFRET) to study the affinity and kinetics of the interaction of several Arabidopsis thaliana ARFs with an IR7 AuxRE. We show that both DBD and PB1 domains of AtARF2 contribute toward DNA binding, and we identify ARF dimer stability as a key parameter in defining affinity and kinetics seen for the DBDs of different AtARFs. Lastly, we derived an analytical solution for a four-state cyclic model that explains both the kinetics and the affinity of the interaction between AtARF2 and IR7. Our work demonstrates that the affinity of ARFs towards composite DNA response elements can be tuned by small changes of their dimerization equilibrium suggesting that this effect has major implications for ARF-mediated transcriptional activity.

biochemistry↗

bioRxiv2021

Rapid auxin-mediated phosphorylation of Myosin regulates trafficking and polarity in Arabidopsis

The signaling molecule auxin controls plant development through a well-known transcriptional mechanism that regulates many genes. However, auxin also triggers cellular responses within seconds or minutes, and mechanisms mediating such fast responses have remained elusive. Here, we identified an ultrafast auxin-mediated protein phosphorylation response in Arabidopsis roots that is largely independent of the canonical TIR1/AFB receptors. Among targets of this novel response are Myosin XI and its adaptor protein MadB2. We show that their auxin-mediated phosphorylation regulates trafficking and polar, subcellular distribution of PIN auxin transporters. This phosphorylation-based auxin signaling module is indispensable during developmental processes that rely on auxin-mediated PIN repolarization, such as termination of shoot gravitropic bending or vasculature formation and regeneration. Hence, we identified a fast, non-canonical auxin response targeting multiple cellular processes and revealed auxin-triggered phosphorylation of a myosin complex as the mechanism for feedback regulation of directional auxin transport, a central component of auxin canalization, which underlies self-organizing plant development.

plant biology↗

bioRxiv2019

Initiation and regulation of vascular tissue identity in the Arabidopsis embryo

Development of plant vascular tissues involves tissue specification, growth, pattern formation and cell type differentiation. While later steps are understood in some detail, it is still largely unknown how the tissue is initially specified. We have used the early Arabidopsis embryo as a simple model to study this process. Using a large collection of marker genes, we find that vascular identity is established in the 16-cell embryo. After a transient precursor state however, there is no persistent uniform tissue identity. Auxin is intimately connected to vascular tissue development. We find that while AUXIN RESPONSE FACTOR5/MONOPTEROS/ (ARF5/MP)-dependent auxin response is required, it is not sufficient for tissue establishment. We therefore used a large-scale enhanced Yeast One Hybrid assay to identify potential regulators of vascular identity. Network and functional analysis of suggest that vascular identity is under robust, complex control. We found that one candidate regulator, the G-class bZIP transcription factor GBF2, modulates vascular gene expression, along with its homolog GBF1. Furthermore, GBFs bind to MP and modulate its activity. Our work uncovers components of a gene regulatory network that controls the initiation of vascular tissue identity, one of which involves the interaction of MP and GBF2 proteins.

plant biology↗

bioRxiv2017

Auxin Response Factors -- output control in auxin biology

The phytohormone auxin is involved in almost all developmental processes in land plants. Most, if not all, of these processes are mediated by changes in gene expression. Auxin acts on gene expression through a short nuclear pathway that converges upon the activation of a family of DNA-binding transcription factors. These AUXIN RESPONSE FACTORS (ARFs) are thus the effector of auxin response and translate the chemical signal to the regulation of a defined set of genes. Given the limited number of dedicated components in auxin signaling, distinct properties among the ARF family likely contributes to the establishment of multiple unique auxin responses in plant development. In the two decades following the identification of the first ARF in Arabidopsis much has been learnt about how these transcription factors act, and how they generate unique auxin responses. Progress in genetics, biochemistry, genomics and structural biology have helped to develop mechanistic models for ARF action. However, despite intensive efforts, many central questions are yet to be addressed. In this review we highlight what has been learnt about ARF transcription factors, and identify outstanding questions and challenges for the near future.

plant biology↗