A deep learning-based toolkit for 3D nuclei segmentation and quantitative analysis in cellular and tissue context.

We present a new set of computational tools that enable accurate and widely applicable 3D segmentation of nuclei in various 3D digital organs. We have developed an approach for ground truth generation and iterative training of 3D nuclear segmentation models, which we applied to popular CellPose, PlantSeg and StarDist algorithms. We provide two high-quality models trained on plant nuclei that enable 3D segmentation of nuclei in datasets obtained from fixed or live samples, acquired from different plant and animal tissues, and stained with various nuclear stains or fluorescent protein-based nuclear reporters. We also share a diverse high-quality training dataset of about 10,000 nuclei. Furthermore, we advanced the MorphoGraphX analysis and visualization software by, among other things, providing a method for linking 3D segmented nuclei to their surrounding cells in 3D digital organs. We found that the nuclear-to-cell volume ratio varies between different ovule tissues and during the development of a tissue. Finally, we extended the PlantSeg 3D segmentation pipeline with a proofreading tool that uses 3D segmented nuclei as seeds to correct cell segmentation errors in difficult-to-segment tissues.

Topological analysis of 3D digital ovules identifies cellular patterns associated with ovule shape diversity.

Tissue morphogenesis remains poorly understood. In plants, a central problem is how the 3D cellular architecture of a developing organ contributes to its final shape. We address this question through a comparative analysis of ovule morphogenesis, taking advantage of the diversity in ovule shape across angiosperms. Here, we provide a 3D digital atlas of Cardamine hirsuta ovule development at single cell resolution and compare it with an equivalent atlas of Arabidopsis thaliana. We introduce nerve-based topological analysis as a tool for unbiased detection of differences in cellular architectures and corroborate identified topological differences between two homologous tissues by comparative morphometrics and visual inspection. We find that differences in topology, cell volume variation and tissue growth patterns in the sheet-like integuments and the bulbous chalaza are associated with differences in ovule curvature. In contrast, the radialized conical ovule primordia and nucelli exhibit similar shapes, despite differences in internal cellular topology and tissue growth patterns. Our results support the notion that the structural organization of a tissue is associated with its susceptibility to shape changes during evolutionary shifts in 3D cellular architecture.

Arabidopsis MCTP family member QUIRKY regulates the formation of the STRUBBELIG receptor kinase complex.

Intercellular communication plays a central role in organogenesis. Tissue morphogenesis in Arabidopsis (Arabidopsis thaliana) requires signaling mediated by a cell surface complex containing the atypical receptor kinase STRUBBELIG (SUB) and the multiple C2 domains and transmembrane region protein QUIRKY (QKY). QKY is required to stabilize SUB at the plasma membrane. However, it is unclear what the in vivo architecture of the QKY/SUB signaling complex is, how it is controlled, and how it relates to the maintenance of SUB at the cell surface. We addressed these questions using a combination of genetics, yeast 2-hybrid assays, and Förster resonance energy transfer (FRET)/fluorescence lifetime imaging microscopy (FLIM) in epidermal cells of seedling roots. We found that QKY promotes the formation of SUB homooligomers in vivo. Homooligomerization of SUB appeared to involve its extracellular domain. We also showed that QKY and SUB physically interact and form a complex at the cell surface in vivo. In addition, the data showed that the N-terminal C2A-B region of QKY interacts with the intracellular domain of SUB. They further revealed that this interaction is essential to maintain SUB levels at the cell surface. Finally, we provided evidence that QKY forms homomultimers in vivo in a SUB-independent manner. We suggest a model in which the physical interaction of QKY with SUB mediates the oligomerization of SUB and attenuates its internalization, thereby maintaining sufficiently high levels of SUB at the cell surface required for the control of tissue morphogenesis.

The annotation and analysis of complex 3D plant organs using 3DCoordX.

A fundamental question in biology concerns how molecular and cellular processes become integrated during morphogenesis. In plants, characterization of 3D digital representations of organs at single-cell resolution represents a promising approach to addressing this problem. A major challenge is to provide organ-centric spatial context to cells of an organ. We developed several general rules for the annotation of cell position and embodied them in 3DCoordX, a user-interactive computer toolbox implemented in the open-source software MorphoGraphX. 3DCoordX enables rapid spatial annotation of cells even in highly curved biological shapes. Using 3DCoordX, we analyzed cellular growth patterns in organs of several species. For example, the data indicated the presence of a basal cell proliferation zone in the ovule primordium of Arabidopsis (Arabidopsis thaliana). Proof-of-concept analyses suggested a preferential increase in cell length associated with neck elongation in the archegonium of Marchantia (Marchantia polymorpha) and variations in cell volume linked to central morphogenetic features of a trap of the carnivorous plant Utricularia (Utricularia gibba). Our work demonstrates the broad applicability of the developed strategies as they provide organ-centric spatial context to cellular features in plant organs of diverse shape complexity.

A Genetic Screen To Identify Genes Influencing the Secondary Redox Couple NADPH/NADP+ in the Yeast Saccharomyces cerevisiae.

NADPH is an important cofactor in the cell. In addition to its role in the biosynthesis of critical metabolites, it plays crucial roles in the regeneration of the reduced forms of glutathione, thioredoxins and peroxiredoxins. The enzymes and pathways that regulate NADPH are thus extremely important to understand, and yet are only partially understood. We have been interested in understanding how NADPH fluxes are altered in the cell. We describe here both an assay and a genetic screen that allows one to discern changes in NADPH levels. The screen exploits the secondary redox property of NADPH. At low levels of glutathione we show that the redox contributions of NADPH become critical for growth, and we have used this to develop a genetic screen for genes affecting NADPH homeostasis. The screen was validated in pathways that both directly (pentose phosphate pathway) and indirectly (glycolytic pathway) affect NADPH levels, and was then exploited to identify mitochondrial genes that affect NADPH homeostasis. A total of 239 mitochondrial gene knockouts were assayed using this screen. Among these, several genes were predicted to play a role in NADPH homeostasis. This included several new genes of unknown function, and others of poorly defined function. We examined two of these genes, FMP40 which encodes a protein required during oxidative stress and GOR1, glyoxylate reductase. Our studies throw new light on these proteins that appear to be major consumers of NADPH in the cell. The genetic screen is thus predicted to be an exceedingly useful tool for investigating NADPH homeostasis.