Anne-Lise Routier-Kierzkowska

Elastic Domains Regulate Growth and Organogenesis in the Plant Shoot Apical Meristem

Shape-Shifting Signals Although orthogonal signaling systems seem to direct various developmental processes, few tissues remain in the same shape as they are at initiation to that of the final form. Arabidopsis leaves are free of the cell migrations that complicate animal development, and thus allowed Kuchen et al. (p. 1092) to track and model the trajectory of leaf growth under a variety of perturbations. Varying the values of parameters in their model produced outputs of different leaf shapes ranging from obcordate, ovate, and oval to elliptic, and offered predictions for genes that regulate the developmental process. The meristem at the growing tip of plants is home to stem cells and is the source of newly differentiating shoots and leaves. New leaves make their first appearance as bulges at the side of the dome-shaped meristem. Although these developmental events are under hormonal control, they also seem to be constrained by the physical properties of the meristem. Kierzkowski et al. (p. 1096) tested physical effects acting on the shoot apical meristem of growing tomato shoots that alter turgor pressure. Again, mathematical modeling combined with observations of plant tissue helped to define the different zones in the meristem that respond to diverse mechanical stimuli. New leaves emerge where they are allowed. Although genetic control of morphogenesis is well established, elaboration of complex shapes requires changes in the mechanical properties of cells. In plants, the first visible sign of leaf formation is a bulge on the flank of the shoot apical meristem. Bulging results from local relaxation of cell walls, which causes them to yield to internal hydrostatic pressure. By manipulation of tissue tension in combination with quantitative live imaging and finite-element modeling, we found that the slow-growing area at the shoot tip is substantially strain-stiffened compared with surrounding fast-growing tissue. We propose that strain stiffening limits growth, restricts organ bulging, and contributes to the meristems functional zonation. Thus, mechanical signals are not just passive readouts of gene action but feed back on morphogenesis.

MorphoGraphX: A platform for quantifying morphogenesis in 4D

Morphogenesis emerges from complex multiscale interactions between genetic and mechanical processes. To understand these processes, the evolution of cell shape, proliferation and gene expression must be quantified. This quantification is usually performed either in full 3D, which is computationally expensive and technically challenging, or on 2D planar projections, which introduces geometrical artifacts on highly curved organs. Here we present MorphoGraphX (www.MorphoGraphX.org), a software that bridges this gap by working directly with curved surface images extracted from 3D data. In addition to traditional 3D image analysis, we have developed algorithms to operate on curved surfaces, such as cell segmentation, lineage tracking and fluorescence signal quantification. The softwares modular design makes it easy to include existing libraries, or to implement new algorithms. Cell geometries extracted with MorphoGraphX can be exported and used as templates for simulation models, providing a powerful platform to investigate the interactions between shape, genes and growth. DOI: http://dx.doi.org/10.7554/eLife.05864.001

Cellular Force Microscopy for in Vivo Measurements of Plant Tissue Mechanics

Although growth and morphogenesis are controlled by genetics, physical shape change in plant tissue results from a balance between cell wall loosening and intracellular pressure. Despite recent work demonstrating a role for mechanical signals in morphogenesis, precise measurement of mechanical properties at the individual cell level remains a technical challenge. To address this challenge, we have developed cellular force microscopy (CFM), which combines the versatility of classical microindentation techniques with the high automation and resolution approaching that of atomic force microscopy. CFM’s large range of forces provides the possibility to map the apparent stiffness of both plasmolyzed and turgid tissue as well as to perform micropuncture of cells using very high stresses. CFM experiments reveal that, within a tissue, local stiffness measurements can vary with the level of turgor pressure in an unexpected way. Altogether, our results highlight the importance of detailed physically based simulations for the interpretation of microindentation results. CFM’s ability to be used both to assess and manipulate tissue mechanics makes it a method of choice to unravel the feedbacks between mechanics, genetics, and morphogenesis.

The pollen tube: a soft shell with a hard core

Plant cell expansion is controlled by a fine-tuned balance between intracellular turgor pressure, cell wall loosening and cell wall biosynthesis. To understand these processes, it is important to gain in-depth knowledge of cell wall mechanics. Pollen tubes are tip-growing cells that provide an ideal system to study mechanical properties at the single cell level. With the available approaches it was not easy to measure important mechanical parameters of pollen tubes, such as the elasticity of the cell wall. We used a cellular force microscope (CFM) to measure the apparent stiffness of lily pollen tubes. In combination with a mechanical model based on the finite element method (FEM), this allowed us to calculate turgor pressure and cell wall elasticity, which we found to be around 0.3 MPa and 20-90 MPa, respectively. Furthermore, and in contrast to previous reports, we showed that the difference in stiffness between the pollen tube tip and the shank can be explained solely by the geometry of the pollen tube. CFM, in combination with an FEM-based model, provides a powerful method to evaluate important mechanical parameters of single, growing cells. Our findings indicate that the cell wall of growing pollen tubes has mechanical properties similar to rubber. This suggests that a fully turgid pollen tube is a relatively stiff, yet flexible cell that can react very quickly to obstacles or attractants by adjusting the direction of growth on its way through the female transmitting tissue.

Morphomechanical Innovation Drives Explosive Seed Dispersal

Summary How mechanical and biological processes are coordinated across cells, tissues, and organs to produce complex traits is a key question in biology. Cardamine hirsuta, a relative of Arabidopsis thaliana, uses an explosive mechanism to disperse its seeds. We show that this trait evolved through morphomechanical innovations at different spatial scales. At the organ scale, tension within the fruit wall generates the elastic energy required for explosion. This tension is produced by differential contraction of fruit wall tissues through an active mechanism involving turgor pressure, cell geometry, and wall properties of the epidermis. Explosive release of this tension is controlled at the cellular scale by asymmetric lignin deposition within endocarp b cells—a striking pattern that is strictly associated with explosive pod shatter across the Brassicaceae plant family. By bridging these different scales, we present an integrated mechanism for explosive seed dispersal that links evolutionary novelty with complex trait innovation. Video Abstract

Measuring the mechanical properties of plant cells by combining micro-indentation with osmotic treatments

Highlight: A combination of osmotic treatments, micro-indentation with cellular force microscopy, and inverse finite-element modelling gives an estimate for both turgor pressure and cell wall elasticity in plant cells.

New stereoscopic reconstruction protocol for scanning electron microscope images and its application to in vivo replicas of the shoot apical meristem

The shoot apical meristem is a small and delicate organ, usually hidden between the young leaves and flowers that it produces. One approach to study meristem geometry and growth consists of taking consecutive replicas from the living meristem surface. In this paper, we present a new stereoscopic reconstruction method for this non-invasive replica protocol, which is applicable to study of growth and geometry of individual cells. This method had been used by the authors to study shoot apical meristem of two species: Arabidopsis thaliana (L.) Heynh. and Anagallis arvensis L., and can be extended to other species and organs. Scanning electron micrographs of the same replica are made at two different angles of view. The obtained stereopairs are used for the dense, three dimensional reconstruction of the replica surface. At the same time, some of the microscope parameters are refined based on the differences between the two micrographs. Three dimensional cell outlines are next extracted from the dense continuous reconstruction, and provide a basis for the quantification of meristem geometry and growth. The new reconstruction protocol can be used with different types of scanning electron microscopes, single- or multi-staged, does not require the identical working distance for the two micrographs of the stereopair, and can be used within a large range of magnifications, corresponding to the cases of either orthogonal or central projection model. It is based largely on recently published algorithms for stereoscopic vision. The reconstruction protocol can be used also for other stereoscopic applications based on scanning electron microscopy. The codes are written in Matlab and are freely available on request to the authors.

Influence of clavata3-2 mutation on early flower development in Arabidopsis thaliana: quantitative analysis of changing geometry

Early development of the flower primordium has been studied in Arabidopsis thaliana clavata3-2 (clv3-2) plants with the aid of sequential in vivo replicas and longitudinal microtome sections. Sequential replicas show that, although there is no regular phyllotaxis in the clv3-2 inflorescence shoot apex, the sites of new primordium formation are, to a large extent, predictable. The primordium always appears in a wedge-like region of the meristem periphery flanked by two older primordia. In general, stages of primordium development in clv3-2 are similar to the wild type, but quantitative geometry analysis shows that the clv3-2 primordium shape is affected even before the CLAVATA/WUSCHEL regulatory network would start to operate in the wild-type primordium. The shape of the youngest primordium in the mutant is more variable than in the wild type. In particular, the shape of the adaxial primordium boundary varies and seems to be related to the shape of the space available for the given primordium formation, suggesting that physical constraints play a significant role in primordium shape determination. The role of physical constraints is also manifested in that the shape of the primordium in the later stages, as well as the number and position of sepals, are adjusted to the available space. Longitudinal sections of clv3-2 apices show that the shape of surface cells of the meristem and young primordium is different from the wild type. Moreover, there is only one tunica layer in both the meristem and in the primordium until it becomes a bulge that is distinctly separated from the meristem. Starting from this stage, the anticlinal divisions predominate in subprotodermal cells, suggesting that the distribution of periclinal and anticlinal cell divisions in the early development of the flower primordium is not directly affected by the clv3-2 mutation.

Why plants make puzzle cells, and how their shape emerges

The shape and function of plant cells are often highly interdependent. The puzzle-shaped cells that appear in the epidermis of many plants are a striking example of a complex cell shape, however their functional benefit has remained elusive. We propose that these intricate forms provide an effective strategy to reduce mechanical stress in the cell wall of the epidermis. When tissue-level growth is isotropic, we hypothesize that lobes emerge at the cellular level to prevent formation of large isodiametric cells that would bulge under the stress produced by turgor pressure. Data from various plant organs and species support the relationship between lobes and growth isotropy, which we test with mutants where growth direction is perturbed. Using simulation models we show that a mechanism actively regulating cellular stress plausibly reproduces the development of epidermal cell shape. Together, our results suggest that mechanical stress is a key driver of cell-shape morphogenesis.

Morphogenesis at the inflorescence shoot apex of Anagallis arvensis: surface geometry and growth in comparison with the vegetative shoot

Quantitative analysis of geometry and surface growth based on the sequential replica method is used to compare morphogenesis at the shoot apex of Anagallis arvensis in the reproductive and vegetative phases of development. Formation of three types of lateral organs takes place at the Anagallis shoot apical meristem (SAM): vegetative leaf primordia are formed during the vegetative phase and leaf-like bracts and flower primordia during the reproductive phase. Although the shapes of all the three types of primordia are very similar during their early developmental stages, areal growth rates and anisotropy of apex surface growth accompanying formation of leaf or bract primordia are profoundly different from those during formation of flower primordia. This provides an example of different modes of de novo formation of a given shape. Moreover, growth accompanying the formation of the boundary between the SAM and flower primordium is entirely different from growth at the adaxial leaf or bract primordium boundary. In the latter, areal growth rates at the future boundary are the lowest of all the apex surface, while in the former they are relatively very high. The direction of maximal growth rate is latitudinal (along the future boundary) in the case of leaf or bract primordium but meridional (across the boundary) in the case of flower. The replica method does not enable direct analysis of growth in the direction perpendicular to the apex surface (anticlinal direction). Nevertheless, the reconstructed surfaces of consecutive replicas taken from an individual apex allow general directions of SAM surface bulging accompanying primordium formation to be recognized. Precise alignment of consecutive reconstructions shows that the direction of initial bulging during the leaf or bract formation is nearly parallel to the shoot axis (upward bulging), while in the case of flower it is perpendicular to the axis (lateral bulging). In future, such 3D reconstructions can be used to assess displacement velocity fields so that growth in the anticlinal direction can be assessed. In terms of self-perpetuation, the inflorescence SAM of Anagallis differs from the SAM in the vegetative phase in that the centrally located region of slow growth is less distinct in the inflorescence SAM. Moreover, the position of this slowly growing zone with respect to cells is not stable in the course of the meristem ontogeny.