The three-dimensional organization of the genome, which strikingly correlates with gene activity, is critical for many cellular processes. The evolution of molecular techniques has allowed us to unveil chromatin structure at an unprecedented resolution. The most intriguing chromatin structures observed in animals are TADs (Topologically Associating Domains), which represent the functional and structural chromatin domains demarcating the genome. Structural proteins such as insulators proteins, on the other hand, have been shown to play crucial roles in mediating the formation of TADs. However, major structural factors relevant to chromatin structure are still waiting to be discovered in land plants. My preliminary work shows that TADs are widely distributed across the rice genome, and motif sequence analysis suggests the enrichment of plant-specific transcription factors at TAD boundaries, which jointly give rise to an exciting hypothesis that these proteins might be the long-sought-after insulators in land plants. By using various state-of-the-art molecular and computational tools, this timely project aims to fill a huge gap in plant functional genomics and substantially advance our understanding of three-dimensional chromatin structure. This project consists four major aims, which collectively will uncover the identities of plant insulator proteins and generate insights into the dynamics of structural chromatin domains during stress adaptation. Aim 1 will identify and characterize the stability and plasticity of functional chromatin domains in the rice genome during temperature stress adaptation. Aim 2 will identify insulator elements and other structural features of chromatin packing in the Marchantia polymorpha genome from a structural genomics approach. Aim 3 will establish the role of candidate proteins as plant insulators. Lastly, Aim 4 will generate functional insights into the molecular mechanism by which plant insulators shape the three-dimensional genome.

During evolution of multicellularity, cells differentiated to become specialized and interdependent. Multicellular organisms invented channels for nutrient exchange and communication between cells. Plants uniquely developed plasmodesmata, complex cell-cell connections traversing the cell wall. Roles ascribed to plasmodesmata include selective transport of signals, ions, metabolites, RNAs and proteins. Due to technical hurdles, composition, structure and regulation of plasmodesmatal conductance remain enigmatic. Genetic approaches to study plasmodesmata were hampered by lethality or redundancy. Novel technologies now set the stage for resolving roles of plasmodesmata in transport and signaling in an interdisciplinary approach. We will use proximity labeling proteomics to obtain plasmodesmatal composition, and PAINT and cryo electron tomography (cryoET) for near atomic structures. Models of plasmodesmata will be built from bottom up and top down approaches and combined with quantitative assessment of plasmodesmatal activity. Novel biosensor approaches together with knock down by genome editing will permit quantitation of transport of the diverse cargo. Single cell sequencing helps fine-tuning mutant selection and targeting of subtypes. Four labs will join forces: highly recognized experts in biophysics and cryoET (WB), advanced imaging and developmental signaling (RS), high-end proteomics and lipidomics (WS), and interactomics, transporters and cutting-edge biosensor technology (WF). We will iteratively address: (1) systematic quantitative identification of components, (2) their localization and dynamics, (3) structures and molecular building blocks of diverse plasmodesmatal types, and (4) transport and signaling mechanisms. We expect breakthrough discoveries and completely new understanding of plasmodesmatal function and evolution. Since plasmodesmata play key roles in nutrient allocation and virus spread, we lay the basis for novel biotech solutions in agriculture.

Europe was the continent where Neanderthal and Anatomically Modern Human (AMH) interacted in a supposed time window of up to five millennia around 40,000 years ago (40 ka BP). The fate of our species versus the demise of Neanderthals, the geospatial timing of this process, the behavioural implications of their interaction, are hotly debated topics in archaeology. In this context, chronology plays a pivotal role, with radiocarbon (14C) dating representing the backbone back to 50 ka BP. However, due to varying atmospheric 14C concentrations in the past, the method requires calibration to an independently dated archive. So far, only the past 14 ka BP can profit from the most direct natural archive of 14C activity, which are annually resolved tree-ring chronologies. Before this time, temporal resolution is remarkably lower and different calibration records disagree, leading to discrepancies in the calibration of up to 2000 years. This project aims to achieve for the first time an accurate and highly resolved chronology back to 50 ka BP, to establish the timing of when AMHs arrived in Europe, their interaction with Neanderthals and the final cause of Neanderthal extinction. The project involves fieldwork in Mediterranean and southeast Europe to find more glacial trees, the study of the existing collection of glacial conifers, exceptional 14C precision for 14C dates in the Glacial, and the cutting-edge methodology in linking floating tree-ring chronologies to 10Be on the ice core scale. The results of this work will be crucial in solving some of the most interesting puzzles in European prehistory. With tree-rings, the resolution will be an order of magnitude higher, and using the most recent advances in the AMS technique, we will obtain confidence intervals of only a few centuries in glacial times. This project will be of pivotal importance for key periods in human evolution.