During early plant embryogenesis, precursors for all major tissues and stem cells are formed. While several components of the regulatory framework are known, how cell fates are instructed by genome-wide transcriptional activity remains unanswered - in part because of difficulties in capturing transcriptome changes at cellular resolution. Here, we have adapted a two-component transgenic labelling system to purify cell type-specific nuclear RNA and generate a transcriptome atlas of early Arabidopsis embryo development, with focus on root stem cell niche formation. We validated the dataset through gene expression analysis, and show that gene activity shifts in a spatio-temporal manner, likely signifying transcriptional reprogramming, to induce developmental processes reflecting cell states and state transitions. This atlas provides the most comprehensive tissue- and cell-specific description of genome-wide gene activity in the early plant embryo, and serves as a valuable resource for understanding the genetic control of early plant development.
Fertilization of flowering plants requires the organization of complex tasks, many of which become integrated by the female gametophyte (FG). The FG is a few-celled haploid structure that orchestrates division of labor to coordinate successful interaction with the sperm cells and their transport vehicle, the pollen tube. As reproductive outcome is directly coupled to evolutionary success, the underlying mechanisms are under robust molecular control, including integrity check and repair mechanisms. Here, we review progress on understanding the development and function of the FG, starting with the functional megaspore, which represents the haploid founder cell of the FG. We highlight recent achievements that have greatly advanced our understanding of pollen tube attraction strategies and the mechanisms that regulate plant hybridization and gamete fusion. In addition, we discuss novel insights into plant polyploidization strategies that expand current concepts on the evolution of flowering plants.
Living eukaryotic cells typically contain large quantities of highly dynamic mitochondria, which sustain the cells’ energy and redox homeostasis. Growing evidence suggests that mitochondria can functionally differ among but also within cells. The extent and biological significance of mitochondrial diversity is still largely unexplored, due to technical limitations that hamper profiling of individual organelles. Previous measurements of the cell’s interior have shown that membrane-bound compartments respond to metabolic manipulation by changes in their surface stiffness, suggesting that mechano-physical properties are a valuable readout of mitochondrial function. We here present the establishment of a robust multi-step analysis pipeline that allows one to profile mechano-physical properties of single mitochondria at the nanoscale using Atomic Force Microscopy (AFM). Firstly, we developed a rapid cell-type specific isolation protocol (mRACE), which selectively functionalizes mitochondria with biotin, facilitating isolation by streptavidin decorated microbeads. We established the technique for human and rat cell cultures, the invertebrate Caenorhabditis elegans, and the model plant Arabidopsis thaliana. Based on this versatile tool, we detected diversity of mitochondrially associated proteins among different tissues, reflecting the trophic condition of the source material. Secondly, a rapid filtration-based mitochondria isolation protocol was established, which was combined with mRACE. Lastly, we established an AFM analysis platform, which generates 3D maps of the nano-topography and mechano-physical properties of individual mitochondria. The comparison of mitochondria with each other revealed an unprecedented diversity in their mechano-physical properties and suggests that shape is not the sole determining parameter for outer membrane stiffness. We expect our results to not only introduce a new dimension for basic mitochondrial research, but in addition to open the door for the exploitation of individual mitochondria for diagnostic characterization.
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