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.

Morphomechanical Innovation Drives Explosive Seed Dispersal.

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.

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.

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.

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.

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.

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.

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

Growth in plants results from the interaction between genetic and signalling networks and the mechanical properties of cells and tissues. There has been a recent resurgence in research directed at understanding the mechanical aspects of growth, and their feedback on genetic regulation. This has been driven in part by the development of new micro-indentation techniques to measure the mechanical properties of plant cells in vivo. However, the interpretation of indentation experiments remains a challenge, since the force measures results from a combination of turgor pressure, cell wall stiffness, and cell and indenter geometry. In order to interpret the measurements, an accurate mechanical model of the experiment is required. Here, we used a plant cell system with a simple geometry, Nicotiana tabacum Bright Yellow-2 (BY-2) cells, to examine the sensitivity of micro-indentation to a variety of mechanical and experimental parameters. Using a finite-element mechanical model, we found that, for indentations of a few microns on turgid cells, the measurements were mostly sensitive to turgor pressure and the radius of the cell, and not to the exact indenter shape or elastic properties of the cell wall. By complementing indentation experiments with osmotic experiments to measure the elastic strain in turgid cells, we could fit the model to both turgor pressure and cell wall elasticity. This allowed us to interpret apparent stiffness values in terms of meaningful physical parameters that are relevant for 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.

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.

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.

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.

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.

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.

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.

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.