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.

Cell-cycle-linked growth reprogramming encodes developmental time into leaf morphogenesis.

How is time encoded into organ growth and morphogenesis? We address this question by investigating heteroblasty, where leaf development and form are modified with progressing plant age. By combining morphometric analyses, fate-mapping through live-imaging, computational analyses, and genetics, we identify age-dependent changes in cell-cycle-associated growth and histogenesis that underpin leaf heteroblasty. We show that in juvenile leaves, cell proliferation competence is rapidly released in a "proliferation burst" coupled with fast growth, whereas in adult leaves, proliferative growth is sustained for longer and at a slower rate. These effects are mediated by the SPL9 transcription factor in response to inputs from both shoot age and individual leaf maturation along the proximodistal axis. SPL9 acts by activating CyclinD3 family genes, which are sufficient to bypass the requirement for SPL9 in the control of leaf shape and in heteroblastic reprogramming of cellular growth. In conclusion, we have identified a mechanism that bridges across cell, tissue, and whole-organism scales by linking cell-cycle-associated growth control to age-dependent changes in organ geometry.

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.

Leaf Morphogenesis: Insights From the Moss Physcomitrium patens.

Specialized photosynthetic organs have appeared several times independently during the evolution of land plants. Phyllids, the leaf-like organs of bryophytes such as mosses or leafy liverworts, display a simple morphology, with a small number of cells and cell types and lack typical vascular tissue which contrasts greatly with flowering plants. Despite this, the leaf structures of these two plant types share many morphological characteristics. In this review, we summarize the current understanding of leaf morphogenesis in the model moss Physcomitrium patens, focusing on the underlying cellular patterns and molecular regulatory mechanisms. We discuss this knowledge in an evolutionary context and identify parallels between moss and flowering plant leaf development. Finally, we propose potential research directions that may help to answer fundamental questions in plant development using moss leaves as a model system.

The Relationship between AGAMOUS and Cytokinin Signaling in the Establishment of Carpeloid Features.

Gynoecium development is dependent on gene regulation and hormonal pathway interactions. The phytohormones auxin and cytokinin are involved in many developmental programs, where cytokinin is normally important for cell division and meristem activity, while auxin induces cell differentiation and organ initiation in the shoot. The MADS-box transcription factor AGAMOUS (AG) is important for the development of the reproductive structures of the flower. Here, we focus on the relationship between AG and cytokinin in Arabidopsis thaliana, and use the weak ag-12 and the strong ag-1 allele. We found that cytokinin induces carpeloid features in an AG-dependent manner and the expression of the transcription factors CRC, SHP2, and SPT that are involved in carpel development. AG is important for gynoecium development, and contributes to regulating, or else directly regulates CRC, SHP2, and SPT. All four genes respond to either reduced or induced cytokinin signaling and have the potential to be regulated by cytokinin via the type-B ARR proteins. We generated a model of a gene regulatory network, where cytokinin signaling is mainly upstream and in parallel with AG activity.

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.

A WOX/Auxin Biosynthesis Module Controls Growth to Shape Leaf Form.

A key challenge in biology is to understand how the regional control of cell growth gives rise to final organ forms. Plant leaves must coordinate growth along both the proximodistal and mediolateral axes to produce their final shape. However, the cell-level mechanisms controlling this coordination remain largely unclear. Here, we show that, in A. thaliana, WOX5, one of the WUSCHEL-RELATED HOMEOBOX (WOX) family of homeobox genes, acts redundantly with WOX1 and WOX3 (PRESSED FLOWER [PRS]) to control leaf shape. Through genetics and hormone measurements, we find that these WOXs act in part through the regional control of YUCCA (YUC) auxin biosynthetic gene expression along the leaf margin. The requirement for WOX-mediated YUC expression in patterning of leaf shape cannot be bypassed by the epidermal expression of YUC, indicating that the precise domain of auxin biosynthesis is important for leaf form. Using time-lapse growth analysis, we demonstrate that WOX-mediated auxin biosynthesis organizes a proximodistal growth gradient that promotes lateral growth and consequently the characteristic ellipsoid A. thaliana leaf shape. We also provide evidence that WOX proteins shape the proximodistal gradient of differentiation by inhibiting differentiation proximally in the leaf blade and promoting it distally. This regulation allows sustained growth of the blade and enables a leaf to attain its final form. In conclusion, we show that the WOX/auxin regulatory module shapes leaf form by coordinating growth along the proximodistal and mediolateral leaf axes.

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.

Quantifying Plant Growth and Cell Proliferation with MorphoGraphX.

Confocal microscopy is widely used to live-image plant tissue. Cell outlines can be visualized using fluorescent probes that mark the cell wall or plasma membrane, enabling the confocal microscope to be used as a 3D scanner with submicron precision. After imaging, the data needs to be analyzed by specialized software to quantify the features of interest, such as cell size and shape, growth rates and anisotropy, and gene expression. Here we present a protocol for the 3D image processing software MorphoGraphX ( www.MorphoGraphX.org ) using time-lapse images of an Arabidopsis thaliana sepal and the shoot apex of tomato.

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.

Global Topological Order Emerges through Local Mechanical Control of Cell Divisions in the Arabidopsis Shoot Apical Meristem.

The control of cell position and division act in concert to dictate multicellular organization in tissues and organs. How these processes shape global order and molecular movement across organs is an outstanding problem in biology. Using live 3D imaging and computational analyses, we extracted networks capturing cellular connectivity dynamics across the Arabidopsis shoot apical meristem (SAM) and topologically analyzed the local and global properties of cellular architecture. Locally generated cell division rules lead to the emergence of global tissue-scale organization of the SAM, facilitating robust global communication. Cells that lie upon more shorter paths have an increased propensity to divide, with division plane placement acting to limit the number of shortest paths their daughter cells lie upon. Cell shape heterogeneity and global cellular organization requires KATANIN, providing a multiscale link between cell geometry, mechanical cell-cell interactions, and global tissue order.

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.

LMI1 homeodomain protein regulates organ proportions by spatial modulation of endoreduplication.

How the interplay between cell- and tissue-level processes produces correctly proportioned organs is a key problem in biology. In plants, the relative size of leaves compared with their lateral appendages, called stipules, varies tremendously throughout development and evolution, yet relevant mechanisms remain unknown. Here we use genetics, live imaging, and modeling to show that in Arabidopsis leaves, the LATE MERISTEM IDENTITY1 (LMI1) homeodomain protein regulates stipule proportions via an endoreduplication-dependent trade-off that limits tissue size despite increasing cell growth. LM1 acts through directly activating the conserved mitosis blocker WEE1, which is sufficient to bypass the LMI1 requirement for leaf proportionality.