Two orthogonal differentiation gradients locally coordinate fruit morphogenesis.

Morphogenesis requires the coordination of cellular behaviors along developmental axes. In plants, gradients of growth and differentiation are typically established along a single longitudinal primordium axis to control global organ shape. Yet, it remains unclear how these gradients are locally adjusted to regulate the formation of complex organs that consist of diverse tissue types. Here we combine quantitative live imaging at cellular resolution with genetics, and chemical treatments to understand the formation of Arabidopsis thaliana female reproductive organ (gynoecium). We show that, contrary to other aerial organs, gynoecium shape is determined by two orthogonal, time-shifted differentiation gradients. An early mediolateral gradient controls valve morphogenesis while a late, longitudinal gradient regulates style differentiation. Local, tissue-dependent action of these gradients serves to fine-tune the common developmental program governing organ morphogenesis to ensure the specialized function of the gynoecium.

Multiple mechanisms behind plant bending.

To survive, plants constantly adapt their body shape to their environment. This often involves remarkably rapid bending of their organs such as stems, leaves and roots. Since plant cells are enclosed by stiff cell walls, they use various strategies for bending their organs, which differ from bending mechanisms of soft animal tissues and involve larger physical forces. Here we attempt to summarize and link different viewpoints on bending mechanisms: genes and signalling, mathematical modelling and biomechanics. We argue that quantifying cell growth and physical forces could open a new level in our understanding of bending and resolve some of its paradoxes.

Cell type-specific dynamics underlie cellular growth variability in plants.

Coordination of growth, patterning and differentiation is required for shaping organs in multicellular organisms. In plants, cell growth is controlled by positional information, yet the behavior of individual cells is often highly heterogeneous. The origin of this variability is still unclear. Using time-lapse imaging, we determined the source and relevance of cellular growth variability in developing organs of Arabidopsis thaliana. We show that growth is more heterogeneous in the leaf blade than in the midrib and petiole, correlating with higher local differences in growth rates between neighboring cells in the blade. This local growth variability coincides with developing stomata. Stomatal lineages follow a specific, time-dependent growth program that is different from that of their surroundings. Quantification of cellular dynamics in the leaves of a mutant lacking stomata, as well as analysis of floral organs, supports the idea that growth variability is mainly driven by stomata differentiation. Thus, the cell-autonomous behavior of specialized cells is the main source of local growth variability in otherwise homogeneously growing tissue. Those growth differences are buffered by the immediate neighbors of stomata and trichomes to achieve robust organ shapes.

Using positional information to provide context for biological image analysis with MorphoGraphX 2.0.

Positional information is a central concept in developmental biology. In developing organs, positional information can be idealized as a local coordinate system that arises from morphogen gradients controlled by organizers at key locations. This offers a plausible mechanism for the integration of the molecular networks operating in individual cells into the spatially coordinated multicellular responses necessary for the organization of emergent forms. Understanding how positional cues guide morphogenesis requires the quantification of gene expression and growth dynamics in the context of their underlying coordinate systems. Here, we present recent advances in the MorphoGraphX software (Barbier de Reuille et al., 2015⁠) that implement a generalized framework to annotate developing organs with local coordinate systems. These coordinate systems introduce an organ-centric spatial context to microscopy data, allowing gene expression and growth to be quantified and compared in the context of the positional information thought to control them.

Live-imaging provides an atlas of cellular growth dynamics in the stamen.

Development of multicellular organisms is a complex process involving precise coordination of growth among individual cells. Understanding organogenesis requires measurements of cellular behaviors over space and time. In plants, such a quantitative approach has been successfully used to dissect organ development in both leaves and external floral organs, such as sepals. However, the observation of floral reproductive organs is hampered as they develop inside tightly closed floral buds, and are therefore difficult to access for imaging. We developed a confocal time-lapse imaging method, applied here to Arabidopsis (Arabidopsis thaliana), which allows full quantitative characterization of the development of stamens, the male reproductive organs. Our lineage tracing reveals the early specification of the filament and the anther. Formation of the anther lobes is associated with a temporal increase of growth at the lobe surface that correlates with intensive growth of the developing locule. Filament development is very dynamic and passes through three distinct phases: (1) initial intense, anisotropic growth, and high cell proliferation; (2) restriction of growth and proliferation to the filament proximal region; and (3) resumption of intense and anisotropic growth, displaced to the distal portion of the filament, without cell proliferation. This quantitative atlas of cellular growth dynamics provides a solid framework for future studies into stamen development.

Mechanochemical feedback mediates tissue bending required for seedling emergence.

Tissue bending is vital to plant development, as exemplified by apical hook formation during seedling emergence by bending of the hypocotyl. How tissue bending is coordinated during development remains poorly understood, especially in plants where cells are attached via rigid cell walls. Asymmetric distribution of the plant hormone auxin underlies differential cell elongation during apical hook formation. Yet the underlying mechanism remains unclear. Here, we demonstrate spatial correlation between asymmetric auxin distribution, methylesterified homogalacturonan (HG) pectin, and mechanical properties of the epidermal layer of the hypocotyl in Arabidopsis. Genetic and cell biological approaches show that this mechanochemical asymmetry is essential for differential cell elongation. We show that asymmetric auxin distribution underlies differential HG methylesterification, and conversely changes in HG methylesterification impact the auxin response domain. Our results suggest that a positive feedback loop between auxin distribution and HG methylesterification underpins asymmetric cell wall mechanochemical properties to promote tissue bending and seedling emergence.

Interplay between Cell Wall and Auxin Mediates the Control of Differential Cell Elongation during Apical Hook Development.

Differential growth plays a crucial role during morphogenesis [1-3]. In plants, development occurs within mechanically connected tissues, and local differences in cell expansion lead to deformations at the organ level, such as buckling or bending [4, 5]. During early seedling development, bending of hypocotyl by differential cell elongation results in apical hook structure that protects the shoot apical meristem from being damaged during emergence from the soil [6, 7]. Plant hormones participate in apical hook development, but not how they mechanistically drive differential growth [8]. Here, we present evidence of interplay between hormonal signals and cell wall in auxin-mediated differential cell elongation using apical hook development as an experimental model. Using genetic and cell biological approaches, we show that xyloglucan (a major primary cell wall component) mediates asymmetric mechanical properties of epidermal cells required for hook development. The xxt1 xxt2 mutant, deficient in xyloglucan [9], displays severe defects in differential cell elongation and hook development. Analysis of xxt1 xxt2 mutant reveals a link between cell wall and transcriptional control of auxin transporters PINFORMEDs (PINs) and AUX1 crucial for establishing the auxin response maxima required for preferential repression of elongation of the cells on the inner side of the hook. Genetic evidence identifies auxin response factor ARF2 as a negative regulator acting downstream of xyloglucan-dependent control of hook development and transcriptional control of polar auxin transport. Our results reveal a crucial feedback process between the cell wall and transcriptional control of polar auxin transport, underlying auxin-dependent control of differential cell elongation in plants.

Panicle Angle is an Important Factor in Tef Lodging Tolerance.

Tef [Eragrostis tef (Zucc.) Trotter] is an important crop in the Horn of Africa, particularly in Ethiopia, where it is a staple food for over 60 million people. However, the productivity of tef remains extremely low in part due to its susceptibility to lodging. Lodging is the displacement of the plant from the upright position, and it is exacerbated by rain, wind and the application of fertilizer. In order to address the issue of global food security, especially in the Horn of Africa, greater insight into the causes of tef lodging is needed. In this study, we combine modeling and biomechanical measurements to compare the properties relating to lodging tolerance in high yielding, improved tef genotypes, and lower yielding natural landraces. Our results indicate that the angle of the panicle contributes to the likelihood of lodging in tef. Varieties with compact panicles and reduced height had increased lodging resistance compared to the other varieties. By comparing different varieties, we found that overall, the landraces of tef lodged less than improved varieties. We constructed a model of stem bending and found that panicle angle was an important determinant of the amount of lodging. The findings from this study provide key information to those involved in tef improvement, especially those interested in lodging tolerance.

Cellular Force Microscopy to Measure Mechanical Forces in Plant Cells.

Cellular force microscopy (CFM) is a noninvasive microindentation method used to measure plant cell stiffness in vivo. CFM is a scanning probe microscopy technique similar in operation to atomic force microscopy (AFM); however, the scale of movement and range of forces are much larger, making it suitable for stiffness measurements on turgid plant cells in whole organs. CFM experiments can be performed on living samples over extended time periods, facilitating the exploration of the dynamics of processes involving mechanics. Different sensor technologies can be used, along with a variety of probe shapes and sizes that can be tailored to specific applications. Measurements can be made for specific indentation depths, forces and timing, allowing for very precise mechanical stimulation of cells with known forces. High forces with sharp tips can also be used for mechanical ablation of cells with force feedback.

Growth and biomechanics of shoot organs.

Plant organs arise through complex interactions between biological and physical factors that control morphogenesis. While there has been tremendous progress in the understanding of the genetics behind development, we know much less about how mechanical forces control growth in plants. In recent years, new multidisciplinary research combining genetics, live-imaging, physics, and computational modeling has begun to fill this gap by revealing the crucial role of biomechanics in the establishment of plant organs. In this review, we provide an overview of our current understanding of growth during initiation, patterning, and expansion of shoot lateral organs. We discuss how growth is controlled by physical forces, and how mechanical stresses generated during growth can control morphogenesis at the level of both cells and tissues. Understanding the mechanical basis of growth and morphogenesis in plants is in its early days, and many puzzling facts are yet to be deciphered.

A Growth-Based Framework for Leaf Shape Development and Diversity.

How do genes modify cellular growth to create morphological diversity? We study this problem in two related plants with differently shaped leaves: Arabidopsis thaliana (simple leaf shape) and Cardamine hirsuta (complex shape with leaflets). We use live imaging, modeling, and genetics to deconstruct these organ-level differences into their cell-level constituents: growth amount, direction, and differentiation. We show that leaf shape depends on the interplay of two growth modes: a conserved organ-wide growth mode that reflects differentiation; and a local, directional mode that involves the patterning of growth foci along the leaf edge. Shape diversity results from the distinct effects of two homeobox genes on these growth modes: SHOOTMERISTEMLESS broadens organ-wide growth relative to edge-patterning, enabling leaflet emergence, while REDUCED COMPLEXITY inhibits growth locally around emerging leaflets, accentuating shape differences created by patterning. We demonstrate the predictivity of our findings by reconstructing key features of C. hirsuta leaf morphology in A. thaliana. VIDEO ABSTRACT.

Cellular basis of growth in plants: geometry matters.

The growth of individual cells underlies the development of biological forms. In plants, cells are interconnected by rigid walls, fixing their position with respect to one another and generating mechanical feedbacks between cells. Current research is shedding new light on how plant growth is controlled by physical inputs at the level of individual cells and growing tissues. In this review, we discuss recent progress in our understanding of the cellular basis of growth from a biomechanical perspective. We describe the role of the cell wall and turgor pressure in growth and highlight the often-overlooked role of cell geometry in this process. It is becoming apparent that a combination of experimental and theoretical approaches is required to answer new emerging questions in the biomechanics of plant morphogenesis. We summarise how this multidisciplinary approach brings us closer to a unified understanding of the generation of biological forms in plants.

The role of APETALA1 in petal number robustness.

Invariant floral forms are important for reproductive success and robust to natural perturbations. Petal number, for example, is invariant in Arabidopsis thaliana flowers. However, petal number varies in the closely related species Cardamine hirsuta, and the genetic basis for this difference between species is unknown. Here we show that divergence in the pleiotropic floral regulator APETALA1 (AP1) can account for the species-specific difference in petal number robustness. This large effect of AP1 is explained by epistatic interactions: A. thaliana AP1 confers robustness by masking the phenotypic expression of quantitative trait loci controlling petal number in C. hirsuta. We show that C. hirsuta AP1 fails to complement this function of A. thaliana AP1, conferring variable petal number, and that upstream regulatory regions of AP1 contribute to this divergence. Moreover, variable petal number is maintained in C. hirsuta despite sufficient standing genetic variation in natural accessions to produce plants with four-petalled flowers.

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.

Clones of cells switch from reduction to enhancement of size variability in Arabidopsis sepals.

Organs form with remarkably consistent sizes and shapes during development, whereas a high variability in growth is observed at the cell level. Given this contrast, it is unclear how such consistency in organ scale can emerge from cellular behavior. Here, we examine an intermediate scale, the growth of clones of cells in Arabidopsis sepals. Each clone consists of the progeny of a single progenitor cell. At early stages, we find that clones derived from a small progenitor cell grow faster than those derived from a large progenitor cell. This results in a reduction in clone size variability, a phenomenon we refer to as size uniformization. By contrast, at later stages of clone growth, clones change their growth pattern to enhance size variability, when clones derived from larger progenitor cells grow faster than those derived from smaller progenitor cells. Finally, we find that, at early stages, fast growing clones exhibit greater cell growth heterogeneity. Thus, cellular variability in growth might contribute to a decrease in the variability of clones throughout the sepal.

Mechanical Shielding of Rapidly Growing Cells Buffers Growth Heterogeneity and Contributes to Organ Shape Reproducibility.

A landmark of developmental biology is the production of reproducible shapes, through stereotyped morphogenetic events. At the cell level, growth is often highly heterogeneous, allowing shape diversity to arise. Yet, how can reproducible shapes emerge from such growth heterogeneity? Is growth heterogeneity filtered out? Here, we focus on rapidly growing trichome cells in the Arabidopsis sepal, a reproducible floral organ. We show via computational modeling that rapidly growing cells may distort organ shape. However, the cortical microtubule alignment along growth-derived maximal tensile stress in adjacent cells would mechanically isolate rapidly growing cells and limit their impact on organ shape. In vivo, we observed such microtubule response to stress and consistently found no significant effect of trichome number on sepal shape in wild-type and lines with trichome number defects. Conversely, modulating the microtubule response to stress in katanin and spiral2 mutant made sepal shape dependent on trichome number, suggesting that, while mechanical signals are propagated around rapidly growing cells, the resistance to stress in adjacent cells mechanically isolates rapidly growing cells, thus contributing to organ shape reproducibility.

Seasonal Regulation of Petal Number.

Four petals characterize the flowers of most species in the Brassicaceae family, and this phenotype is generally robust to genetic and environmental variation. A variable petal number distinguishes the flowers of Cardamine hirsuta from those of its close relative Arabidopsis (Arabidopsis thaliana), and allelic variation at many loci contribute to this trait. However, it is less clear whether C. hirsuta petal number varies in response to seasonal changes in environment. To address this question, we assessed whether petal number responds to a suite of environmental and endogenous cues that regulate flowering time in C. hirsuta We found that petal number showed seasonal variation in C. hirsuta, such that spring flowering plants developed more petals than those flowering in summer. Conditions associated with spring flowering, including cool ambient temperature, short photoperiod, and vernalization, all increased petal number in C. hirsuta Cool temperature caused the strongest increase in petal number and lengthened the time interval over which floral meristems matured. We performed live imaging of early flower development and showed that floral buds developed more slowly at 15°C versus 20°C. This extended phase of floral meristem formation, coupled with slower growth of sepals at 15°C, produced larger intersepal regions with more space available for petal initiation. In summary, the growth and maturation of floral buds is associated with variable petal number in C. hirsuta and responds to seasonal changes in ambient temperature.

On the micro-indentation of plant cells in a tissue context.

The effect of geometry on cell stiffness measured with micro-indentation techniques has been explored in single cells, however it is unclear if results on single cells can be readily transferred to indentation experiments performed on a tissue in vivo. Here we explored this question by using simulation models of osmotic treatments and micro-indentation experiments on 3D multicellular tissues with the finite element method. We found that the cellular context does affect measured cell stiffness, and that several cells of context in each direction are required for optimal results. We applied the model to micro-indentation data obtained with cellular force microscopy on the sepal of A. thaliana, and found that differences in measured stiffness could be explained by cellular geometry, and do not necessarily indicate differences in cell wall material properties or turgor pressure.

Variable Cell Growth Yields Reproducible OrganDevelopment through Spatiotemporal Averaging.

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.