Periodic Reporting for period 3 - SymPore (Plasmodesmata, Symplasmic pores for plant cell-to-cell communication)
Periodo di rendicontazione: 2024-04-01 al 2025-09-30
During the evolution of multicellularity, cells differentiated to become specialized and interdependent. Multicellular organisms invented channels for intracellular nutrient exchange and communication. The plant lineage developed plasmodesmata, complex cell-cell connections that traverse the cell wall and have roles in selective transport of signals, ions, metabolites, RNAs and proteins.
Improving crop yield is of major relevance for food security. Plasmodesmata are thought to play critical roles in many traits important for the productivity and sustainability of crops, e.g. the allocation of carbohydrates from leaves to seeds, flowering time, dormancy, pathogen defense and development. Knowledge of structure and function of plasmodesmata is, therefore, essential for rational improvements of crop yield. Due to technical hurdles, composition, structure and regulation of plasmodesmatal conductance have remained largely enigmatic. Genetic approaches to study plasmodesmata were hampered by lethality or redundancy. However, novel technologies now set the stage for resolving the roles of plasmodesmata in transport and signaling in an interdisciplinary approach. Four labs joined forces: W. Baumeister (Max Planck Institute Biochemistry, Munich; biophysics and cryoET), R. Simon (Heinrich Heine University, Düsseldorf; advanced imaging and developmental signaling), W. Schulze (University of Hohenheim; high-end proteomics and lipidomics), and WB. Frommer (Heinrich Heine University, Düsseldorf; interactomics, transporters and biosensor technology). We have begun to iteratively address: (1) we established the systematic quantitative identification of components using enrichment of plasmodesmata followed by lipidomics and proteomics, (2) we successfully and systematically localize plasmodesmatal protein candidates and analyze the dynamics, (3) we obtained structures and molecular building blocks of diverse plasmodesmatal types, in particular by Cryo electron tomography, and (4) established and used transport and signaling assays to characterize mutants in plasmodesmatal proteins.
Because plasmodesmata are critically involved in many fundamental plant processes, especially crop yield, new insights into structure, function and regulation are critical. The impact on the society is envisaged at several levels - gain in fundamental knowledge, training of students and scientists, and we attempt to develop new ways to adapt crop plants to climate change and increase yield in a sustainable manner.
Improving crop yield is of major relevance for food security. Plasmodesmata are thought to play critical roles in many traits important for the productivity and sustainability of crops, e.g. the allocation of carbohydrates from leaves to seeds, flowering time, dormancy, pathogen defense and development. Knowledge of structure and function of plasmodesmata is, therefore, essential for rational improvements of crop yield. Due to technical hurdles, composition, structure and regulation of plasmodesmatal conductance have remained largely enigmatic. Genetic approaches to study plasmodesmata were hampered by lethality or redundancy. However, novel technologies now set the stage for resolving the roles of plasmodesmata in transport and signaling in an interdisciplinary approach. Four labs joined forces: W. Baumeister (Max Planck Institute Biochemistry, Munich; biophysics and cryoET), R. Simon (Heinrich Heine University, Düsseldorf; advanced imaging and developmental signaling), W. Schulze (University of Hohenheim; high-end proteomics and lipidomics), and WB. Frommer (Heinrich Heine University, Düsseldorf; interactomics, transporters and biosensor technology). We have begun to iteratively address: (1) we established the systematic quantitative identification of components using enrichment of plasmodesmata followed by lipidomics and proteomics, (2) we successfully and systematically localize plasmodesmatal protein candidates and analyze the dynamics, (3) we obtained structures and molecular building blocks of diverse plasmodesmatal types, in particular by Cryo electron tomography, and (4) established and used transport and signaling assays to characterize mutants in plasmodesmatal proteins.
Because plasmodesmata are critically involved in many fundamental plant processes, especially crop yield, new insights into structure, function and regulation are critical. The impact on the society is envisaged at several levels - gain in fundamental knowledge, training of students and scientists, and we attempt to develop new ways to adapt crop plants to climate change and increase yield in a sustainable manner.
Over the first 4 years, we assembled a strong interdisciplinary team and initiated close collaborations between the teams. In moss, dense arrays of plasmodesmatal proteins were detected at interfaces between protonemata at comparatively high densities of >10 plasmodesmata μm−2.
We initiated analyses by cryo-electron tomography (cryoET), however, plant tissues were more challenging subjects for cryoET when comparing the efficacy relative to the well-established work in other organisms by the Baumeister team. Initial experiments using protonemata from the moss P. patens provided first data by cryoET. Due to the presence of ice, structures had to be interpreted with caution. Reproductive organs of A. thaliana and N. benthamiana and trichomes were explored as suitable tissues with small cell sizes. We were developed a workflow for high quality cryoET of P. patents, Arabidopsis and Limonium bicolor. Detailed protocols and data were published in Elife (Pöge et al., 2025). The work also shows subtomogram averaging of plastidic compartments. A second manuscript is currently under review in which unique structures were identified on the surface of the desmotublues.
Proteomic protocols for plasmodesmata enrichment were established and plasmodesmata fractions were analyzed. We identified 870 proteins with large overlap of 422 A. thaliana orthologs in plasmodesmata fractions from P. patens and A. thaliana (PDdb: http://pddb.uni-hohenheim.de(si apre in una nuova finestra)).
In a joint effort, >150 candidate proteins from the plasmodesmata proteome were validated by expressing them as fusions with fluorescent protein (FP) tags and analyzing them using confocal microscopy. We identified >40 novel proteins that preferentially localize to plasmodesmata (Gombos et al. 2023). This result further showed a conservation of targeting mechanism between moss and tobacco.
Notably, we identified a whole series of nuclear pore proteins in the plasmodesmata-enriched fractions. We found also that GFP fusions of the nuclear pore proteins can be identified at plasmodesmata, mutations in nuclear pore proteins led to reduced intercellular transport of proteins. While we cannot exclude artifacts, the data are consistent with the presence of a phase-separated domain in plasmodesmata (Ejike et al., 2025 J. Exp. Bot, accepted; Schladt et al., 2024). We currently explore the potential phase separation that has features analogous to that involved in nuclear transport using independent alternative approaches. We established protein proximity labeling and explored the proxisome of plasmodesmata-localized proteins. To obtain a reliable, near complete inventory of the plasmodesmata, we iteratively improve purification protocols using information gained from these integrated proteomics-imaging approaches. Single cell sequencing and molecular cartography are used to determine the composition of different types of plasmodesmata present in specialized cells (Kim et al., 2021). We established lipid extraction protocols and are optimizing strategies to study lipid-protein interactions. Based on our first interactome and lipid-protein interaction results we functionally analyze loss-of-function mutants for 20 putative plasmodesmata components. We established over seven distinct transport assays (Drop N See, particle bombardment with DNA constructs, microinjection of fluorescent dyes or GFP, as well as miRNA and SHR3 transport assays, calcium biosensor lines) that are being used to evaluate the effect of mutations in plasmodesmatal proteins. About 100 candidate mutant lines are currently under investigation.
We initiated analyses by cryo-electron tomography (cryoET), however, plant tissues were more challenging subjects for cryoET when comparing the efficacy relative to the well-established work in other organisms by the Baumeister team. Initial experiments using protonemata from the moss P. patens provided first data by cryoET. Due to the presence of ice, structures had to be interpreted with caution. Reproductive organs of A. thaliana and N. benthamiana and trichomes were explored as suitable tissues with small cell sizes. We were developed a workflow for high quality cryoET of P. patents, Arabidopsis and Limonium bicolor. Detailed protocols and data were published in Elife (Pöge et al., 2025). The work also shows subtomogram averaging of plastidic compartments. A second manuscript is currently under review in which unique structures were identified on the surface of the desmotublues.
Proteomic protocols for plasmodesmata enrichment were established and plasmodesmata fractions were analyzed. We identified 870 proteins with large overlap of 422 A. thaliana orthologs in plasmodesmata fractions from P. patens and A. thaliana (PDdb: http://pddb.uni-hohenheim.de(si apre in una nuova finestra)).
In a joint effort, >150 candidate proteins from the plasmodesmata proteome were validated by expressing them as fusions with fluorescent protein (FP) tags and analyzing them using confocal microscopy. We identified >40 novel proteins that preferentially localize to plasmodesmata (Gombos et al. 2023). This result further showed a conservation of targeting mechanism between moss and tobacco.
Notably, we identified a whole series of nuclear pore proteins in the plasmodesmata-enriched fractions. We found also that GFP fusions of the nuclear pore proteins can be identified at plasmodesmata, mutations in nuclear pore proteins led to reduced intercellular transport of proteins. While we cannot exclude artifacts, the data are consistent with the presence of a phase-separated domain in plasmodesmata (Ejike et al., 2025 J. Exp. Bot, accepted; Schladt et al., 2024). We currently explore the potential phase separation that has features analogous to that involved in nuclear transport using independent alternative approaches. We established protein proximity labeling and explored the proxisome of plasmodesmata-localized proteins. To obtain a reliable, near complete inventory of the plasmodesmata, we iteratively improve purification protocols using information gained from these integrated proteomics-imaging approaches. Single cell sequencing and molecular cartography are used to determine the composition of different types of plasmodesmata present in specialized cells (Kim et al., 2021). We established lipid extraction protocols and are optimizing strategies to study lipid-protein interactions. Based on our first interactome and lipid-protein interaction results we functionally analyze loss-of-function mutants for 20 putative plasmodesmata components. We established over seven distinct transport assays (Drop N See, particle bombardment with DNA constructs, microinjection of fluorescent dyes or GFP, as well as miRNA and SHR3 transport assays, calcium biosensor lines) that are being used to evaluate the effect of mutations in plasmodesmatal proteins. About 100 candidate mutant lines are currently under investigation.
This highly challenging project provides training of undergraduate students, graduate students and postdoctoral scientists in cutting edge technologies. The new technologies developed by the SymPore team will be made available to the broad scientific community, thereby advancing science broadly. Due to the overall genetic similarity of organisms, insights gained from studying plasmodesmata in the model species used here likely has relevance for crop plants and may in addition provide new perspectives on intercellular bridges in other organisms including fungi, animals and humans. Given that the project will be successful, the knowledge gained will likely provide new possibilities to improve crop yield and climate change resilience of crop plants. Potential applications include the development of new markers for marker-assisted breeding. The approach could be exploited to generate crop plants with improved yield potential, increased yield in stress conditions, resilience to climate change impact, control over flowering time and dormancy to enable earlier or extended planting periods, improved stress tolerance, in particular pathogen resistance and improved nutrient efficiency. Such plants will be important to mitigate the impact of climate change and provide improved food security as well as improved sustainability.