Stiffness transitions in new walls post-cell division differ between Marchantia polymorpha gemmae and Arabidopsis thaliana leaves.

Plant morphogenesis is governed by the mechanics of the cell wall-a stiff and thin polymeric box that encloses the cells. The cell wall is a highly dynamic composite material. New cell walls are added during cell division. As the cells continue to grow, the properties of cell walls are modulated to undergo significant changes in shape and size without breakage. Spatial and temporal variations in cell wall mechanical properties have been observed. However, how they relate to cell division remains an outstanding question. Here, we combine time-lapse imaging with local mechanical measurements via atomic force microscopy to systematically map the cell wall's age and growth, with their stiffness. We make use of two systems, Marchantia polymorpha gemmae, and Arabidopsis thaliana leaves. We first characterize the growth and cell division of M. polymorpha gemmae. We then demonstrate that cell division in M. polymorpha gemmae results in the generation of a temporary stiffer and slower-growing new wall. In contrast, this transient phenomenon is absent in A. thaliana leaves. We provide evidence that this different temporal behavior has a direct impact on the local cell geometry via changes in the junction angle. These results are expected to pave the way for developing more realistic plant morphogenetic models and to advance the study into the impact of cell division on tissue growth.

Tissue folding at the organ-meristem boundary results in nuclear compression and chromatin compaction.

Artificial mechanical perturbations affect chromatin in animal cells in culture. Whether this is also relevant to growing tissues in living organisms remains debated. In plants, aerial organ emergence occurs through localized outgrowth at the periphery of the shoot apical meristem, which also contains a stem cell niche. Interestingly, organ outgrowth has been proposed to generate compression in the saddle-shaped organ-meristem boundary domain. Yet whether such growth-induced mechanical stress affects chromatin in plant tissues is unknown. Here, by imaging the nuclear envelope in vivo over time and quantifying nucleus deformation, we demonstrate the presence of active nuclear compression in that domain. We developed a quantitative pipeline amenable to identifying a subset of very deformed nuclei deep in the boundary and in which nuclei become gradually narrower and more elongated as the cell contracts transversely. In this domain, we find that the number of chromocenters is reduced, as shown by chromatin staining and labeling, and that the expression of linker histone H1.3 is induced. As further evidence of the role of forces on chromatin changes, artificial compression with a MicroVice could induce the ectopic expression of H1.3 in the rest of the meristem. Furthermore, while the methylation status of chromatin was correlated with nucleus deformation at the meristem boundary, such correlation was lost in the h1.3 mutant. Altogether, we reveal that organogenesis in plants generates compression that is able to have global effects on chromatin in individual cells.

Cytoskeletal organization in isolated plant cells under geometry control.

The cytoskeleton plays a key role in establishing robust cell shape. In animals, it is well established that cell shape can also influence cytoskeletal organization. Cytoskeletal proteins are well conserved between animal and plant kingdoms; nevertheless, because plant cells exhibit major structural differences to animal cells, the question arises whether the plant cytoskeleton also responds to geometrical cues. Recent numerical simulations predicted that a geometry-based rule is sufficient to explain the microtubule (MT) organization observed in cells. Due to their high flexural rigidity and persistence length of the order of a few millimeters, MTs are rigid over cellular dimensions and are thus expected to align along their long axis if constrained in specific geometries. This hypothesis remains to be tested in cellulo Here, we explore the relative contribution of geometry to the final organization of actin and MT cytoskeletons in single plant cells of Arabidopsis thaliana We show that the cytoskeleton aligns with the long axis of the cells. We find that actin organization relies on MTs but not the opposite. We develop a model of self-organizing MTs in three dimensions, which predicts the importance of MT severing, which we confirm experimentally. This work is a first step toward assessing quantitatively how cellular geometry contributes to the control of cytoskeletal organization in living plant cells.

It's about time: Analysing simplifying assumptions for modelling multi-step pathways in systems biology.

Thoughtful use of simplifying assumptions is crucial to make systems biology models tractable while still representative of the underlying biology. A useful simplification can elucidate the core dynamics of a system. A poorly chosen assumption can, however, either render a model too complicated for making conclusions or it can prevent an otherwise accurate model from describing experimentally observed dynamics. Here, we perform a computational investigation of sequential multi-step pathway models that contain fewer pathway steps than the system they are designed to emulate. We demonstrate when such models will fail to reproduce data and how detrimental truncation of a pathway leads to detectable signatures in model dynamics and its optimised parameters. An alternative assumption is suggested for simplifying such pathways. Rather than assuming a truncated number of pathway steps, we propose to use the assumption that the rates of information propagation along the pathway is homogeneous and, instead, letting the length of the pathway be a free parameter. We first focus on linear pathways that are sequential and have first-order kinetics, and we show how this assumption results in a three-parameter model that consistently outperforms its truncated rival and a delay differential equation alternative in recapitulating observed dynamics. We then show how the proposed assumption allows for similarly terse and effective models of non-linear pathways. Our results provide a foundation for well-informed decision making during model simplifications.

Nitrate modulates stem cell dynamics in Arabidopsis shoot meristems through cytokinins.

The shoot apical meristem (SAM) is responsible for the generation of all the aerial parts of plants. Given its critical role, dynamical changes in SAM activity should play a central role in the adaptation of plant architecture to the environment. Using quantitative microscopy, grafting experiments, and genetic perturbations, we connect the plant environment to the SAM by describing the molecular mechanism by which cytokinins signal the level of nutrient availability to the SAM. We show that a systemic signal of cytokinin precursors mediates the adaptation of SAM size and organogenesis rate to the availability of mineral nutrients by modulating the expression of WUSCHEL, a key regulator of stem cell homeostasis. In time-lapse experiments, we further show that this mechanism allows meristems to adapt to rapid changes in nitrate concentration, and thereby modulate their rate of organ production to the availability of mineral nutrients within a few days. Our work sheds light on the role of the stem cell regulatory network by showing that it not only maintains meristem homeostasis but also allows plants to adapt to rapid changes in the environment.

The self-organization of plant microtubules inside the cell volume yields their cortical localization, stable alignment, and sensitivity to external cues.

Many cell functions rely on the ability of microtubules to self-organize as complex networks. In plants, cortical microtubules are essential to determine cell shape as they guide the deposition of cellulose microfibrils, and thus control mechanical anisotropy of the cell wall. Here we analyze how, in turn, cell shape may influence microtubule behavior. Building upon previous models that confined microtubules to the cell surface, we introduce an agent model of microtubules enclosed in a three-dimensional volume. We show that the microtubule network has spontaneous aligned configurations that could explain many experimental observations without resorting to specific regulation. In particular, we find that the preferred cortical localization of microtubules emerges from directional persistence of the microtubules, and their interactions with each other and with the stiff wall. We also identify microtubule parameters that seem relatively insensitive to cell shape, such as length or number. In contrast, microtubule array anisotropy depends on local curvature of the cell surface and global orientation follows robustly the longest axis of the cell. Lastly, we find that geometric cues may be overcome, as the network is capable of reorienting toward weak external directional cues. Altogether our simulations show that the microtubule network is a good transducer of weak external polarity, while at the same time, easily reaching stable global configurations.

Cell size and growth regulation in the Arabidopsis thaliana apical stem cell niche.

Cell size and growth kinetics are fundamental cellular properties with important physiological implications. Classical studies on yeast, and recently on bacteria, have identified rules for cell size regulation in single cells, but in the more complex environment of multicellular tissues, data have been lacking. In this study, to characterize cell size and growth regulation in a multicellular context, we developed a 4D imaging pipeline and applied it to track and quantify epidermal cells over 3-4 d in Arabidopsis thaliana shoot apical meristems. We found that a cell size checkpoint is not the trigger for G2/M or cytokinesis, refuting the unexamined assumption that meristematic cells trigger cell cycle phases upon reaching a critical size. Our data also rule out models in which cells undergo G2/M at a fixed time after birth, or by adding a critical size increment between G2/M transitions. Rather, cell size regulation was intermediate between the critical size and critical increment paradigms, meaning that cell size fluctuations decay by ∼75% in one generation compared with 100% (critical size) and 50% (critical increment). Notably, this behavior was independent of local cell-cell contact topologies and of position within the tissue. Cells grew exponentially throughout the first >80% of the cell cycle, but following an asymmetrical division, the small daughter grew at a faster exponential rate than the large daughter, an observation that potentially challenges present models of growth regulation. These growth and division behaviors place strong constraints on quantitative mechanistic descriptions of the cell cycle and growth control.

WUSCHEL protein movement mediates stem cell homeostasis in the Arabidopsis shoot apex.

WUSCHEL (WUS) is a homeodomain transcription factor produced in cells of the niche/organizing center (OC) of shoot apical meristems. WUS specifies stem cell fate and also restricts its own levels by activating a negative regulator, CLAVATA3 (CLV3), in adjacent cells of the central zone (CZ). Here we show that the WUS protein, after being synthesized in cells of the OC, migrates into the CZ, where it activates CLV3 transcription by binding to its promoter elements. Using a computational model, we show that maintenance of the WUS gradient is essential to regulate stem cell number. Migration of a stem cell-inducing transcription factor into adjacent cells to activate a negative regulator, thereby restricting its own accumulation, is a theme that is unique to plant stem cell niches.

A continuous growth model for plant tissue.

Morphogenesis in plants and animals involves large irreversible deformations. In plants, the response of the cell wall material to internal and external forces is determined by its mechanical properties. An appropriate model for plant tissue growth must include key features such as anisotropic and heterogeneous elasticity and cell dependent evaluation of mechanical variables such as turgor pressure, stress and strain. In addition, a growth model needs to cope with cell divisions as a necessary part of the growth process. Here we develop such a growth model, which is capable of employing not only mechanical signals but also morphogen signals for regulating growth. The model is based on a continuous equation for updating the resting configuration of the tissue. Simultaneously, material properties can be updated at a different time scale. We test the stability of our model by measuring convergence of growth results for a tissue under the same mechanical and material conditions but with different spatial discretization. The model is able to maintain a strain field in the tissue during re-meshing, which is of particular importance for modeling cell division. We confirm the accuracy of our estimations in two and three-dimensional simulations, and show that residual stresses are less prominent if strain or stress is included as input signal to growth. The approach results in a model implementation that can be used to compare different growth hypotheses, while keeping residual stresses and other mechanical variables updated and available for feeding back to the growth and material properties.

Stress and strain provide positional and directional cues in development.

The morphogenesis of organs necessarily involves mechanical interactions and changes in mechanical properties of a tissue. A long standing question is how such changes are directed on a cellular scale while being coordinated at a tissular scale. Growing evidence suggests that mechanical cues are participating in the control of growth and morphogenesis during development. We introduce a mechanical model that represents the deposition of cellulose fibers in primary plant walls. In the model both the degree of material anisotropy and the anisotropy direction are regulated by stress anisotropy. We show that the finite element shell model and the simpler triangular biquadratic springs approach provide equally adequate descriptions of cell mechanics in tissue pressure simulations of the epidermis. In a growing organ, where circumferentially organized fibers act as a main controller of longitudinal growth, we show that the fiber direction can be correlated with both the maximal stress direction and the direction orthogonal to the maximal strain direction. However, when dynamic updates of the fiber direction are introduced, the mechanical stress provides a robust directional cue for the circumferential organization of the fibers, whereas the orthogonal to maximal strain model leads to an unstable situation where the fibers reorient longitudinally. Our investigation of the more complex shape and growth patterns in the shoot apical meristem where new organs are initiated shows that a stress based feedback on fiber directions is capable of reproducing the main features of in vivo cellulose fiber directions, deformations and material properties in different regions of the shoot. In particular, we show that this purely mechanical model can create radially distinct regions such that cells expand slowly and isotropically in the central zone while cells at the periphery expand more quickly and in the radial direction, which is a well established growth pattern in the meristem.

Plant stem cell maintenance involves direct transcriptional repression of differentiation program.

In animal systems, master regulatory transcription factors (TFs) mediate stem cell maintenance through a direct transcriptional repression of differentiation promoting TFs. Whether similar mechanisms operate in plants is not known. In plants, shoot apical meristems serve as reservoirs of stem cells that provide cells for all above ground organs. WUSCHEL, a homeodomain TF produced in cells of the niche, migrates into adjacent cells where it specifies stem cells. Through high-resolution genomic analysis, we show that WUSCHEL represses a large number of genes that are expressed in differentiating cells including a group of differentiation promoting TFs involved in leaf development. We show that WUS directly binds to the regulatory regions of differentiation promoting TFs; KANADI1, KANADI2, ASYMMETRICLEAVES2 and YABBY3 to repress their expression. Predictions from a computational model, supported by live imaging, reveal that WUS-mediated repression prevents premature differentiation of stem cell progenitors, being part of a minimal regulatory network for meristem maintenance. Our work shows that direct transcriptional repression of differentiation promoting TFs is an evolutionarily conserved logic for stem cell regulation.

The MOSS Physcomitrella patens reproductive organ development is highly organized, affected by the two SHI/STY genes and by the level of active auxin in the SHI/STY expression domain.

In order to establish a reference for analysis of the function of auxin and the auxin biosynthesis regulators SHORT INTERNODE/STYLISH (SHI/STY) during Physcomitrella patens reproductive development, we have described male (antheridial) and female(archegonial) development in detail, including temporal and positional information of organ initiation. This has allowed us to define discrete stages of organ morphogenesis and to show that reproductive organ development in P. patens is highly organized and that organ phyllotaxis differs between vegetative and reproductive development. Using the PpSHI1 and PpSHI2 reporter and knockout lines, the auxin reporters GmGH3(pro):GUS and PpPINA(pro):GFP-GUS, and the auxin-conjugating transgene PpSHI2(pro):IAAL, we could show that the PpSHI genes, and by inference also auxin, play important roles for reproductive organ development in moss. The PpSHI genes are required for the apical opening of the reproductive organs, the final differentiation of the egg cell, and the progression of canal cells into a cell death program. The apical cells of the archegonium, the canal cells, and the egg cell are also sites of auxin responsiveness and are affected by reduced levels of active auxin, suggesting that auxin mediates PpSHI function in the reproductive organs.

Xyloglucan deficiency leads to a reduction in turgor pressure and changes in cell wall properties, affecting early seedling establishment.

Xyloglucan is believed to play a significant role in cell wall mechanics of dicot plants. Surprisingly, Arabidopsis plants defective in xyloglucan biosynthesis exhibit nearly normal growth and development. We investigated a mutant line, cslc-Δ5, lacking activity in all five Arabidopsis cellulose synthase like-C (CSLC) genes responsible for xyloglucan backbone biosynthesis. We observed that this xyloglucan-deficient line exhibited reduced cellulose crystallinity and increased pectin levels, suggesting the existence of feedback mechanisms that regulate wall composition to compensate for the absence of xyloglucan. These alterations in cell wall composition in the xyloglucan-absent plants were further linked to a decrease in cell wall elastic modulus and rupture stress, as observed through atomic force microscopy (AFM) and extensometer-based techniques. This raised questions about how plants with such modified cell wall properties can maintain normal growth. Our investigation revealed two key factors contributing to this phenomenon. First, measurements of turgor pressure, a primary driver of plant growth, revealed that cslc-Δ5 plants have reduced turgor, preventing the compromised walls from bursting while still allowing growth to occur. Second, we discovered the conservation of elastic asymmetry (ratio of axial to transverse wall elasticity) in the mutant, suggesting an additional mechanism contributing to the maintenance of normal growth. This novel feedback mechanism between cell wall composition and mechanical properties, coupled with turgor pressure regulation, plays a central role in the control of plant growth and is critical for seedling establishment in a mechanically challenging environment by affecting shoot emergence and root penetration.

Alignment between PIN1 polarity and microtubule orientation in the shoot apical meristem reveals a tight coupling between morphogenesis and auxin transport.

Morphogenesis during multicellular development is regulated by intercellular signaling molecules as well as by the mechanical properties of individual cells. In particular, normal patterns of organogenesis in plants require coordination between growth direction and growth magnitude. How this is achieved remains unclear. Here we show that in Arabidopsis thaliana, auxin patterning and cellular growth are linked through a correlated pattern of auxin efflux carrier localization and cortical microtubule orientation. Our experiments reveal that both PIN1 localization and microtubule array orientation are likely to respond to a shared upstream regulator that appears to be biomechanical in nature. Lastly, through mathematical modeling we show that such a biophysical coupling could mediate the feedback loop between auxin and its transport that underlies plant phyllotaxis.

Mean-field theory approach to three-dimensional nematic phase transitions in microtubules.

Microtubules are dynamic intracellular fibers that have been observed experimentally to undergo spontaneous self-alignment. We formulate a three-dimensional (3D) mean-field theory model to analyze the nematic phase transition of microtubules growing and interacting within a 3D space, then make a comparison with computational simulations. We identify a control parameter G_{eff} and predict a unique critical value G_{eff}=1.56 for which a phase transition can occur. Furthermore, we show both analytically and using simulations that this predicted critical value does not depend on the presence of zippering. The mean-field theory developed here provides an analytical estimate of microtubule patterning characteristics without running time-consuming simulations and is a step towards bridging scales from microtubule behavior to multicellular simulations.

A randomized trial comparing rates of abdominal contamination and postoperative infection among natural orifice transluminal endoscopic surgery, laparoscopic surgery, and open surgery in pigs.

BACKGROUND: Bacterial contamination of the abdominal cavity and infectious complications have been debated concerning transgastric natural orifice transluminal endoscopic surgery (NOTES) procedures. OBJECTIVE: The aim of this study was to compare bacterial contamination of the abdominal cavity and clinically relevant infections after open, laparoscopic, and transgastric NOTES procedures. DESIGN: Randomized survival study in a porcine model. SETTING: Animal laboratory at a university hospital. INTERVENTION: Thirty pigs were randomized to open, laparoscopic, or transgastric NOTES uterine horn resection under sterile conditions. Bacterial cultures were obtained from the pelvic area immediately at entry of the abdominal cavity and just before closure. The left uterine horn was dissected and ligated. The animals survived for 4 weeks. At necropsy, bacterial culture was obtained from the pelvic area. MAIN OUTCOME MEASUREMENTS: Perioperative: operation time and incision length, bacterial growth in abdominal samples. Postoperative: infections or complications, weight gain. Necropsy: signs of peritonitis or infection, abdominal bacterial growth. RESULTS: Procedure time was significantly longer for transgastric NOTES. At the start of the procedure, 4 of the NOTES animals showed positive cultures, but only 1 showed positive cultures at the end. No open surgery or laparoscopic surgery animals showed positive cultures at these time points. At necropsy, none of the animals in the NOTES group showed bacterial growth, whereas 4 open surgery animals and 3 laparoscopic surgery animals had positive cultures. Four of these animals (2 from each group) had concurrent wound infections. LIMITATIONS: Small sample size and lack of power calculation. CONCLUSION: This study indicates that clinically relevant infections are rare after transgastric NOTES procedures despite evidence of bacterial contamination and longer operating times.

High-Throughput 3D Phenotyping of Plant Shoot Apical Meristems From Tissue-Resolution Data.

Confocal imaging is a well-established method for investigating plant phenotypes on the tissue and organ level. However, many differences are difficult to assess by visual inspection and researchers rely extensively on ad hoc manual quantification techniques and qualitative assessment. Here we present a method for quantitatively phenotyping large samples of plant tissue morphologies using triangulated isosurfaces. We successfully demonstrate the applicability of the approach using confocal imaging of aerial organs in Arabidopsis thaliana. Automatic identification of flower primordia using the surface curvature as an indication of outgrowth allows for high-throughput quantification of divergence angles and further analysis of individual flowers. We demonstrate the throughput of our method by quantifying geometric features of 1065 flower primordia from 172 plants, comparing auxin transport mutants to wild type. Additionally, we find that a paraboloid provides a simple geometric parameterisation of the shoot inflorescence domain with few parameters. We utilise parameterisation methods to provide a computational comparison of the shoot apex defined by a fluorescent reporter of the central zone marker gene CLAVATA3 with the apex defined by the paraboloid. Finally, we analyse the impact of mutations which alter mechanical properties on inflorescence dome curvature and compare the results with auxin transport mutants. Our results suggest that region-specific expression domains of genes regulating cell wall biosynthesis and local auxin transport can be important in maintaining the wildtype tissue shape. Altogether, our results indicate a general approach to parameterise and quantify plant development in 3D, which is applicable also in cases where data resolution is limited, and cell segmentation not possible. This enables researchers to address fundamental questions of plant development by quantitative phenotyping with high throughput, consistency and reproducibility.

Context-specific functions of transcription factors controlling plant development: From leaves to flowers.

Plant development is regulated by transcription factors that often act in more than one process and stage of development. Yet the molecular mechanisms that govern the functional diversity and specificity of these proteins remains far from understood. Flower development provides an ideal context to study these mechanisms since the development of distinct floral organs depends on similar but distinct combinations of transcriptional regulators. Recent work also highlights the importance of leaf polarity regulators as additional key factors in flower initiation, floral organ morphogenesis, and possibly floral organ positioning. A detailed understanding of how these factors work in combination will enable us to address outstanding questions in flower development including how distinct shapes and positions of floral organs are generated. Experimental approaches and computer-based modeling will be required to characterize gene-regulatory networks at the level of single cells.

A 3D gene expression atlas of the floral meristem based on spatial reconstruction of single nucleus RNA sequencing data.

Cellular heterogeneity in growth and differentiation results in organ patterning. Single-cell transcriptomics allows characterization of gene expression heterogeneity in developing organs at unprecedented resolution. However, the original physical location of the cell is lost during this methodology. To recover the original location of cells in the developing organ is essential to link gene activity with cellular identity and function in plants. Here, we propose a method to reconstruct genome-wide gene expression patterns of individual cells in a 3D flower meristem by combining single-nuclei RNA-seq with microcopy-based 3D spatial reconstruction. By this, gene expression differences among meristematic domains giving rise to different tissue and organ types can be determined. As a proof of principle, the method is used to trace the initiation of vascular identity within the floral meristem. Our work demonstrates the power of spatially reconstructed single cell transcriptome atlases to understand plant morphogenesis. The floral meristem 3D gene expression atlas can be accessed at http://threed-flower-meristem.herokuapp.com .