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 software's 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

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 meristem's functional zonation. Thus, mechanical signals are not just passive readouts of gene action but feed back on morphogenesis.T he plant shoot apical meristem is composed of two regions, the slow-growing central region, which contains the stem cell niche, and the surrounding periphery, where cells divide rapidly and new organs are initiated (1-4). New organ primordia initiate at accumulation points of the plant hormone auxin (5-7). In addition to triggering gene regulatory pathways, auxin induces cell wall acidification (8), which increases expansin activity (9) that modifies cross-links in the cell wall matrix. Disruption of auxin signaling suppresses organ initiation, which can be restored by the local application of auxin (7,10,11). Bulging in the meristem flank can also be triggered by local cell wall loosening with expansin (12, 13) or pectin methyl-esterase (PME) (14, 15). These bulges can develop into normal organs, which suggests that a mechanical signal is involved in primordium differentiation. Additional support for mechanical signals in this pathway comes from the recent hypothesis that stress in the cell wall is the signal that orients the microtubule network and the PIN-FORMED 1 (PIN1) auxin transporter (16,17). Yet despite the accumulating evidence for an instructive role for mechanical signals in organogenesis, the mechanical properties of the shoot apex have only recently begun to be explored (15, 18). Here, we examine both the elastic and plastic properties of the shoot apex and link them to growth dynamics.Tomato vegetative shoot apices were imaged at 11-hour intervals by confocal microscopy in order to monitor their growth. Images were analyzed with MorphoGraphX (19) (Fig. 1) to compute relative changes in cell surface area (Fig. 2).Cell surface expansion was 25% on average in the central region and between 45 and 80% on average in the periphery, depending on the stage of development of the adjacent primordium. The boundary region between the primordium and the meristem displayed little growth. Our data closely resembled growth patterns in other species (1-4).In order to examine meristem material properties, we induced tissue deformation by manipulating turgor pressure with osmotic treatments using mannitol and NaCl. Experiments started by adapting the samples in solutions of 0.2 M osmotica...

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.

Organ sizes and shapes are strikingly reproducible, despite the variable growth and division of individual cells within them. To reveal which mechanisms enable this precision, we designed a screen for disrupted sepal size and shape uniformity in Arabidopsis and identified mutations in the mitochondrial i-AAA protease FtsH4. Counterintuitively, through live imaging we observed that variability of neighboring cell growth was reduced in ftsh4 sepals. We found that regular organ shape results from spatiotemporal averaging of the cellular variability in wild-type sepals, which is disrupted in the less-variable cells of ftsh4 mutants. We also found that abnormal, increased accumulation of reactive oxygen species (ROS) in ftsh4 mutants disrupts organ size consistency. In wild-type sepals, ROS accumulate in maturing cells and limit organ growth, suggesting that ROS are endogenous signals promoting termination of growth. Our results demonstrate that spatiotemporal averaging of cellular variability is required for precision in organ size.

How organs reach their final shape is a central yet unresolved question in developmental biology. Here we investigate whether mechanical cues contribute to this process. We analyze the epidermal cells of the Arabidopsis sepal, focusing on cortical microtubule arrays, which align along maximal tensile stresses and restrict growth in that direction through their indirect impact on the mechanical anisotropy of cell walls. We find a good match between growth and microtubule orientation throughout most of the development of the sepal. However, at the sepal tip, where organ maturation initiates and growth slows down in later stages, microtubules remain in a configuration consistent with fast anisotropic growth, i.e., transverse, and the anisotropy of their arrays even increases. To understand this apparent paradox, we built a continuous mechanical model of a growing sepal. The model demonstrates that differential growth in the sepal can generate transverse tensile stress at the tip. Consistently, microtubules respond to mechanical perturbations and align along maximal tension at the sepal tip. Including this mechanical feedback in our growth model of the sepal, we predict an impact on sepal shape that is validated experimentally using mutants with either increased or decreased microtubule response to stress. Altogether, this suggests that a mechanical feedback loop, via microtubules acting both as stress sensor and growth regulator, channels the growth and shape of the sepal tip. We propose that this proprioception mechanism is a key step leading to growth arrest in the whole sepal in response to its own growth.