Geert De Jaeger

Membrane contact sites (MCSs) are evolutionarily conserved intracellular nanodomains that physically bridge opposing lipid bilayers to facilitate non-vesicular communication and maintain cellular homeostasis. In plants, endoplasmic reticulum-plasma membrane (ER-PM) contact sites play fundamental roles in environmental adaptation, and are populated by specialized proteins which act as tethers such as Synaptotagmin 1 (SYT1). However, a comprehensive view of the molecular machinery governing processes at these junctions is still needed. In this work, we integrate affinity purification mass spectrometry, TurboID proximity labeling, and a dual-localization reanalysis of HyperLOPIT spatial proteomics to functionally map the protein interaction landscape of the ER-PM contact sites protein SYT1. Beyond recovering established ER-PM MCS functions, our analysis identified uncharacterized proteins as bona fide resident components of these junctions, and revealed that these nanodomains act as docking platforms that anchor the monolignol biosynthetic complex. By spatially organizing Membrane Steroid Binding Proteins and cytochrome P450 enzymes, our findings support a model where SYT1-mediated anchoring of this metabolon to ER-PM contact sites optimizes monolignol export required for stress-induced lignification. Ultimately, this proteomic framework expands the functional repertoire of ER-PM contact sites, opening new avenues to uncover hidden roles of MCSs across diverse eukaryotic systems.

Membrane contact sites (MCSs) are evolutionarily conserved intracellular nanodomains that physically bridge opposing lipid bilayers to facilitate non-vesicular communication and maintain cellular homeostasis. In plants, endoplasmic reticulum-plasma membrane (ER-PM) contact sites play fundamental roles in environmental adaptation, and are populated by specialized proteins which act as tethers such as Synaptotagmin 1 (SYT1). However, a comprehensive view of the molecular machinery governing processes at these junctions is still needed. In this work, we integrate affinity purification mass spectrometry, TurboID proximity labeling, and a dual-localization reanalysis of HyperLOPIT spatial proteomics to functionally map the protein interaction landscape of the ER-PM contact sites protein SYT1. Beyond recovering established ER-PM MCS functions, our analysis identified uncharacterized proteins as bona fide resident components of these junctions, and revealed that these nanodomains act as docking platforms that anchor the monolignol biosynthetic complex. By spatially organizing Membrane Steroid Binding Proteins and cytochrome P450 enzymes, our findings support a model where SYT1-mediated anchoring of this metabolon to ER-PM contact sites optimizes monolignol export required for stress-induced lignification. Ultimately, this proteomic framework expands the functional repertoire of ER-PM contact sites, opening new avenues to uncover hidden roles of MCSs across diverse eukaryotic systems.

: Callose synthase is responsible for the targeted deposition of the β-1,3-glucan polymer, callose which underlines essential plant developmental processes, including cell division, pathogen defense or cell-cell communication. The architecture of the callose synthase complex (CALSC) as well as the molecular mechanisms of callose synthesis remain unknown. Here we report an integrative characterisation of the Arabidopsis CALS complex, with the most enriched subunits, CALS1, CALS2 and CALS3, forming its core. Structurally, CALSC assembles into a trimer, requiring the plant-specific Bag domain to mediate inter-subunit associations. The biological importance of CALSC assembly is highlighted by the simultaneous loss of CALS1 and CALS3, which abolishes plasmodesmal callose deposition and affects symplastic transport. Site-directed mutagenesis and molecular dynamics simulations depict the topology of the CALS1 active site in detail, including the components of the enzymatic reaction. We pinpoint the translocating tunnel through which the nascent glucan is delivered and mechanistically confirm the role of transmembrane helix 8 in regulating glucan export. Our work provides unprecedented insight into the molecular architecture of the CALSC and the distinct changes from maturation to activity at the plasma membrane, while showcasing the mechanisms involved in callose synthesis at the molecular level.

Genomic integrity is constantly challenged by transcription/replication conflicts, a major source of replication stress and instability across all life forms. While extensive studies have elucidated mechanisms for resolving transcription/replication conflicts in animals, yeast, and prokaryotes, their counterparts in plants remain largely unexplored. Through a forward genetic screen, we identified LUMINIDEPENDENS (LD), previously known for its role in regulating the flowering repressor FLC , as a key factor in mitigating replication stress in plants. Notably, transcriptomic analyses reveal that LD loss results in the upregulation of over half of the Arabidopsis genes, placing LD as a global transcriptional repressor. Consistent with this role, LD directly binds a substantial portion of the Arabidopsis genome and interacts with the MED18 subunit of the Mediator complex to modulate RNA polymerase II phosphorylation. These findings uncover a fundamental function of LD in fine-tuning transcription at a genome-wide scale, with potentially an additional role in suppressing transcription–replication conflicts by locally dampening transcription and promoting replication fork progression. Our work highlights an intriguing genome-protective strategy in plants, that could shed light on mechanisms involved in transcription–replication conflict management in eukaryotic systems.

In eukaryotes, chromosome segregation relies on attachment to the spindle, ensured by the kinetochore. The outer kinetochore attaches to the microtubules and is named after three sub-complexes KNL1C, MIS12C, and NDC80C (KMN). While the KMN complex comprises ten proteins in humans S. cerevisiae , its conservation in more distant eukaryotes is unclear. Here, we aimed to define the KMN complex in the plant Arabidopsis using affinity purification and identified thirteen KMN proteins. Seven were previously known to have a conserved function (atMIS12, atNNF1, atNDC80, atSPC24, atSPC25, atNUF2, and atKNL1) and six were uncharacterized. These six proteins show remote similarity to yeast/human KMN-associated proteins, whose homologs have not yet been characterized in plants. We named them atDSN1, atCSM1, atNSL1.1/.2, and atZWINT1.1/.2. We confirmed kinetochore localization for atDSN1, atCSM1, atNSL1.1, and atZWINT1.1 in planta . In addition, atDSN1 , atCSM1 , and ZWINT1.1/.2 are essential, further supporting their kinetochore function. AlphaFold3 predicts an alike3D organization of the KMN complex in plants and mammals. We conclude that the KMN complex is globally conserved with a matching composition and similar organization in distant eukaryotes, with some local variations, suggesting its presence in the common ancestors of all living eukaryotes.

Plant root development is tightly regulated and depends on the coordination among different cell types located in the root stem cell niche (SCN). A complex network of regulators includes the gasotransmitter nitric oxide (NO) known to inhibit primary root growth, modify root architecture and alter meristem organisation. However, little is known about the molecular targets of this signalling molecule during root SCN homeostasis. Here, genetic, molecular and physiological evidence support that TGA transcription factors (TFs) of the bZIP family, and specifically PERIANTHIA (PAN), contribute to the regulation of the root SCN through NO sensing. This gasotransmitter induces reversible S-nitrosylation of PAN, thereby inhibiting its binding to DNA and associated transcriptional activation; whilst the protein-SNO reductase activity of TRXh5 can denitrosylate PAN. This switch on and off mechanism for PAN activity modulates gene expression in the SCN and the functioning of the quiescent centre, thereby altering columella stem cell maintenance through a NO-dependent mechanism. Furthermore, PAN interacts selectively with several TGA members which are susceptible to differential S-nitrosylation. Given the functional redundancy within the TGA family, analysis of quadruple and pentuple mutants revealed close coordination between PAN and other members of this family of TFs, which contributes to the NO sensing during the development of the root apical meristem. Collectively, these findings reveal a molecular framework in which the TGA family, primarily coordinated by PAN activity, acts as a master NO sensor in the root SCN.

Plants frequently encounter hyperosmotic stress due to drought and salinity, leading to rapid water loss, reduced turgor pressure, and decreased cell volume. This contraction drastically alters plasma membrane tension, a physical parameter that must be strictly maintained to support mechanosignaling and cell expansion. However, the mechanisms by which plants adjust their membrane surface area to match a shrinking cell volume remain poorly understood. Here, we identify selective autophagy dependent degradation of plasma membrane-derived clathrin-coated vesicles (CCVs) in response to hyperosmotic shock. This pathway involves the recruitment of the endocytic TPLATE complex (TPC) to autophagosomes in an osmotic-stress dependent manner. Through correlative light and electron microscopy (CLEM) and electron tomography (ET), we provide ultrastructural evidence of the physical association of CCVs with autophagosome membranes. These autophagosomes contain endocytic machinery, including TPC and clathrin, and are targeted to the vacuole. Mechanistically, we show that the conserved ATG8-interacting motifs (AIMs) in the AtEH1/Pan1 and AtEH2/Pan1 TPC subunits interact with ATG8, suggesting that they facilitate the recruitment of CCVs to autophagosomes. Using time-lapse imaging, we demonstrate that the acute induction of autophagy is precisely coupled to the reduction in cell volume under hyperosmolar conditions. Our results suggest that endocytic removal of excess plasma membrane to maintain membrane tension and cellular integrity is coupled to TPC-mediated CCV-phagy. These findings reveal a homeostatic mechanism that enables plants to adapt to the challenges of drought and salinity. Significance Statement: All living cells must maintain the physical integrity of their outer membrane. How cells can adapt their surface area to respond to rapid changes in cell volume, such as those caused by drought or salt stress, remains a fundamental question in biology. We identify a mechanism in plants involving autophagy – a cellular recycling pathway – where plasma membrane-derived vesicles are targeted for degradation following salt or osmotic stress. This pathway involves the TPLATE complex, an evolutionary ancient and essential endocytic complex in plants, which directly interacts with the autophagy protein ATG8. This discovery reveals how plants adapt to osmotic stress by modulating their membrane properties, supporting a framework for future improvement of crop resilience in response to salinity and drought.

Eukaryotic cells maintain homeostasis of their outer membrane by controlled internalization of lipid and protein constituents via endocytosis¹. Endocytosis is evolutionary conserved and utilizes similar structural folds. How these structural folds are combined into proteins and protein complexes however differs between eukaryotic kingdoms². The TPLATE complex in plants is an evolutionary ancient protein module that combines several endocytic folds into a single octameric protein complex^3–5. Its molecular architecture, lipid-nucleated condensate formation, and its requirement for clathrin cage curvature revealed its function in endocytosis initiation in plants^6–8. Mechanistic understanding of how this complex drives membrane deformation during plant endocytosis is, however, lacking. Here, we used an integrative structural approach to obtain a precise molecular structure of the TPLATE complex. In addition, our approach allowed visualizing the structural flexibility that hallmarks this enigmatic complex. We prove that the intrinsic structural flexibility is required for its functionality and membrane recruitment. The membrane binding interface consists of several domains with differential lipid preferences. Finally, we show that the crescent shape of the structured part of the complex is sufficient for membrane curvature generation. Our mechanistic insight answers the long-standing question of how plants execute endocytosis without cytoskeletal-based force generation.