Self-organization underlies developmental robustness in plants.

Development is a self-organized process that builds on cells and their interactions. Cells are heterogeneous in gene expression, growth, and division; yet how development is robust despite such heterogeneity is a fascinating question. Here, we review recent progress on this topic, highlighting how developmental robustness is achieved through self-organization. We will first discuss sources of heterogeneity, including stochastic gene expression, heterogeneity in growth rate and direction, and heterogeneity in division rate and precision. We then discuss cellular mechanisms that buffer against such noise, including Paf1C- and miRNA-mediated denoising, spatiotemporal growth averaging and compensation, mechanisms to improve cell division precision, and coordination of growth rate and developmental timing between different parts of an organ. We also discuss cases where such heterogeneity is not buffered but utilized for development. Finally, we highlight potential directions for future studies of noise and developmental robustness.

Growth couples temporal and spatial fluctuations of tissue properties during morphogenesis.

Living tissues display fluctuations-random spatial and temporal variations of tissue properties around their reference values-at multiple scales. It is believed that such fluctuations may enable tissues to sense their state or their size. Recent theoretical studies developed specific models of fluctuations in growing tissues and predicted that fluctuations of growth show long-range correlations. Here, we elaborated upon these predictions and we tested them using experimental data. We first introduced a minimal model for the fluctuations of any quantity that has some level of temporal persistence or memory, such as concentration of a molecule, local growth rate, or mechanical property. We found that long-range correlations are generic, applying to any such quantity, and that growth couples temporal and spatial fluctuations, through a mechanism that we call "fluctuation stretching"-growth enlarges the length scale of variation of this quantity. We then analyzed growth data from sepals of the model plant Arabidopsis and we quantified spatial and temporal fluctuations of cell growth using the previously developed cellular Fourier transform. Growth appears to have long-range correlations. We compared different genotypes and growth conditions: mutants with lower or higher response to mechanical stress have lower temporal correlations and longer-range spatial correlations than wild-type plants. Finally, we used theoretical predictions to merge experimental data from all conditions and developmental stages into a unifying curve, validating the notion that temporal and spatial fluctuations are coupled by growth. Altogether, our work reveals kinematic constraints on spatiotemporal fluctuations that have an impact on the robustness of morphogenesis.

What Is a Plant Cell Type in the Age of Single-Cell Biology? It's Complicated.

One of the fundamental questions in developmental biology is how a cell is specified to differentiate as a specialized cell type. Traditionally, plant cell types were defined based on their function, location, morphology, and lineage. Currently, in the age of single-cell biology, researchers typically attempt to assign plant cells to cell types by clustering them based on their transcriptomes. However, because cells are dynamic entities that progress through the cell cycle and respond to signals, the transcriptome also reflects the state of the cell at a particular moment in time, raising questions about how to define a cell type. We suggest that these complexities and dynamics of cell states are of interest and further consider the roles signaling, stochasticity, cell cycle, and mechanical forces play in plant cell fate specification. Once established, cell identity must also be maintained. With the wealth of single-cell data coming out, the field is poised to elucidate both the complexity and dynamics of cell states.

Plant Membrane-On-A-Chip: A Platform for Studying Plant Membrane Proteins and Lipids.

The cell plasma membrane is a two-dimensional, fluid mosaic material composed of lipids and proteins that create a semipermeable barrier defining the cell from its environment. Compared with soluble proteins, the methodologies for the structural and functional characterization of membrane proteins are challenging. An emerging tool for studies of membrane proteins in mammalian systems is a "plasma membrane on a chip," also known as a supported lipid bilayer. Here, we create the "plant-membrane-on-a-chip,″ a supported bilayer made from the plant plasma membranes of Arabidopsis thaliana, Nicotiana benthamiana, or Zea mays. Membrane vesicles from protoplasts containing transgenic membrane proteins and their native lipids were incorporated into supported membranes in a defined orientation. Membrane vesicles fuse and orient systematically, where the cytoplasmic side of the membrane proteins faces the chip surface and constituents maintain mobility within the membrane plane. We use plant-membrane-on-a-chip to perform fluorescent imaging to examine protein-protein interactions and determine the protein subunit stoichiometry of FLOTILLINs. We report here that like the mammalian FLOTILLINs, FLOTILLINs expressed in Arabidopsis form a tetrameric complex in the plasma membrane. This plant-membrane-on-a-chip approach opens avenues to studies of membrane properties of plants, transport phenomena, biophysical processes, and protein-protein and protein-lipid interactions in a convenient, cell-free platform.

An optimized live imaging and growth analysis approach for Arabidopsis Sepals.

Background: Arabidopsis thaliana sepals are excellent models for analyzing growth of entire organs due to their relatively small size, which can be captured at a cellular resolution under a confocal microscope [1]. To investigate how growth of different tissue layers generates unique organ morphologies, it is necessary to live-image deep into the tissue. However, imaging deep cell layers of the sepal is practically challenging, as it is hindered by the presence of extracellular air spaces between mesophyll cells, among other factors which causes optical aberrations. Image processing is also difficult due to the low signal-to-noise ratio of the deeper tissue layers, an issue mainly associated with live imaging datasets. Addressing some of these challenges, we provide an optimized methodology for live imaging sepals and subsequent image processing. This helps us track the growth of individual cells on the outer and inner epidermal layers, which are the key drivers of sepal morphogenesis. Results: For live imaging sepals across all tissue layers at early stages of development, we found that the use of a bright fluorescent membrane marker, coupled with increased laser intensity and an enhanced Z- resolution produces high-quality images suitable for downstream image processing. Our optimized parameters allowed us to image the bottommost cell layer of the sepal (inner epidermal layer) without compromising viability. We used a 'voxel removal' technique to visualize the inner epidermal layer in MorphoGraphX [2, 3] image processing software. Finally, we describe the process of optimizing the parameters for creating a 2.5D mesh surface for the inner epidermis. This allowed segmentation and parent tracking of individual cells through multiple time points, despite the weak signal of the inner epidermal cells. Conclusion: We provide a robust pipeline for imaging and analyzing growth across inner and outer epidermal layers during early sepal development. Our approach can potentially be employed for analyzing growth of other internal cell layers of the sepals as well. For each of the steps, approaches, and parameters we used, we have provided in-depth explanations to help researchers understand the rationale and replicate our pipeline.

Growth directions and stiffness across cell layers determine whether tissues stay smooth or buckle.

From smooth to buckled, nature exhibits organs of various shapes and forms. How cellular growth patterns produce smooth organ shapes such as leaves and sepals remains unclear. Here we show that unidirectional growth and comparable stiffness across both epidermal layers of Arabidopsis sepals are essential for smoothness. We identified a mutant with ectopic ASYMMETRIC LEAVES 2 (AS2) expression on the outer epidermis. Our analysis reveals that ectopic AS2 expression causes outer epidermal buckling at early stages of sepal development, due to conflicting growth directions and unequal epidermal stiffnesses. Aligning growth direction and increasing stiffness of the outer epidermis restores smoothness. Furthermore, buckling influences auxin efflux transporter protein PIN-FORMED 1 polarity to generate outgrowth in the later stages, suggesting that buckling is sufficient to initiate outgrowths. Our findings suggest that in addition to molecular cues influencing tissue mechanics, tissue mechanics can also modulate molecular signals, giving rise to well-defined shapes.

Tradeoff Between Speed and Robustness in Primordium Initiation Mediated by Auxin-CUC1 Interaction.

Robustness is the reproducible development of a phenotype despite stochastic noise. It often involves tradeoffs with other performance metrics, but the mechanisms underlying such tradeoffs were largely unknown. An Arabidopsis flower robustly develops four sepals from four precisely positioned auxin maxima. The development related myb-like 1 (drmy1) mutant generates stochastic noise in auxin signaling that disrupts both the robust position and number of sepal primordia. Here, we found that increased expression of CUP-SHAPED COTYLEDON1 (CUC1), a boundary specification transcription factor, in the drmy1 mutant underlies this loss of robustness. CUC1 surrounds and amplifies stochastic auxin patches in drmy1 to form variably positioned auxin maxima and sepal primordia. Removing CUC1 from drmy1 provides time for the noise in auxin signaling to resolve into four precisely positioned auxin maxima, restoring robust sepal initiation. However, removing CUC1 decreases auxin maxima intensity and slows down sepal initiation. Thus, CUC1 increases morphogenesis speed but impairs robustness against auxin noise. Further, using a computational model, we found that the observed phenotype can be explained by the effect of CUC1 in repolarizing PIN FORMED1 (PIN1), a polar auxin transporter. Thus, our study illustrates a tradeoff between speed and robustness during development.

The dynamics and biophysics of shape formation: Common themes in plant and animal morphogenesis.

The emergence of tissue form in multicellular organisms results from the complex interplay between genetics and physics. In both plants and animals, cells must act in concert to pattern their behaviors. Our understanding of the factors sculpting multicellular form has increased dramatically in the past few decades. From this work, common themes have emerged that connect plant and animal morphogenesis-an exciting connection that solidifies our understanding of the developmental basis of multicellular life. In this review, we will discuss the themes and the underlying principles that connect plant and animal morphogenesis, including the coordination of gene expression, signaling, growth, contraction, and mechanical and geometric feedback.

A 3-component module maintains sepal flatness in Arabidopsis.

As in origami, morphogenesis in living systems heavily relies on tissue curving and folding, through the interplay between biochemical and biomechanical cues. In contrast, certain organs maintain their flat posture over several days. Here we identified a pathway, which is required for the maintenance of organ flatness, taking the sepal, the outermost floral organ, in Arabidopsis as a model system. Through genetic, cellular and mechanical approaches, our results demonstrate that global gene expression regulator VERNALIZATION INDEPENDENCE 4 (VIP4) fine-tunes the mechanical properties of sepal cell walls and maintains balanced growth on both sides of the sepals, mainly by orchestrating the distribution pattern of AUXIN RESPONSE FACTOR 3 (ARF3). vip4 mutation results in softer cell walls and faster cell growth on the adaxial sepal side, which eventually cause sepals to bend outward. Downstream of VIP4, ARF3 works through modulating auxin signaling to down-regulate pectin methylesterase VANGUARD1, resulting in decreased cell wall stiffness. Our work unravels a 3-component module, which relates hormonal patterns to organ curvature, and actively maintains sepal flatness during its growth.

Growth couples temporal and spatial fluctuations of tissue properties during morphogenesis.

Living tissues display fluctuations - random spatial and temporal variations of tissue properties around their reference values - at multiple scales. It is believed that such fluctuations may enable tissues to sense their state or their size. Recent theoretical studies developed specific models of fluctuations in growing tissues and predicted that fluctuations of growth show long-range correlations. Here we elaborated upon these predictions and we tested them using experimental data. We first introduced a minimal model for the fluctuations of any quantity that has some level of temporal persistence or memory, such as concentration of a molecule, local growth rate, or mechanical properties. We found that long-range correlations are generic, applying to to any such quantity, and that growth couples temporal and spatial fluctuations. We then analysed growth data from sepals of the model plant Arabidopsis and we quantified spatial and temporal fluctuations of cell growth using the previously developed Cellular Fourier Transform. Growth appears to have long-range correlations. We compared different genotypes and growth conditions: mutants with altered response to mechanical stress have lower temporal correlations and longer-range spatial correlations than wild-type plants. Finally, we used a theoretical prediction to collapse experimental data from all conditions and developmental stages, validating the notion that temporal and spatial fluctuations are coupled by growth. Altogether, our work reveals kinematic constraints on spatiotemporal fluctuations that have an impact on the robustness of morphogenesis.

Robust organ size in Arabidopsis is primarily governed by cell growth rather than cell division patterns.

Organ sizes and shapes are highly reproducible, or robust, within a species and individuals. Arabidopsis thaliana sepals, which are the leaf-like organs that enclose flower buds, have consistent size and shape, which indicates robust development. Counterintuitively, variability in cell growth rate over time and between cells facilitates robust development because cumulative cell growth averages to a uniform rate. Here we investigate how sepal morphogenesis is robust to changes in cell division but not robust to changes in cell growth variability. We live image and quantitatively compare the development of sepals with increased or decreased cell division rate (lgo mutant and LGO overexpression, respectively), a mutant with altered cell growth variability (ftsh4), and double mutants combining these. We find that robustness is preserved when cell division rate changes because there is no change in the spatial pattern of growth. Meanwhile when robustness is lost in ftsh4 mutants, cell growth accumulates unevenly, and cells have disorganized growth directions. Thus, we demonstrate in vivo that both cell growth rate and direction average in robust development, preserving robustness despite changes in cell division.

The ratio of auxin to cytokinin controls leaf development and meristem initiation in Physcomitrium patens.

Crosstalk between auxin and cytokinin contributes to widespread developmental processes, including root and shoot meristem maintenance, phyllotaxy, and vascular patterning. However, our understanding of crosstalk between these hormones is limited primarily to angiosperms. The moss Physcomitrium patens (formerly Physcomitrella patens) is a powerful system for studying plant hormone function. Auxin and cytokinin play similar roles in regulating moss gametophore (shoot) architecture, to those in flowering plant shoots. However, auxin-cytokinin crosstalk is poorly understood in moss. Here we find that the ratio of auxin to cytokinin is an important determinant of development in P. patens, especially during leaf development and branch stem cell initiation. Addition of high levels of auxin to P. patens gametophores blocks leaf outgrowth. However, simultaneous addition of high levels of both auxin and cytokinin partially restores leaf outgrowth, suggesting that the ratio of these hormones is the predominant factor. Likewise, during branch initiation and outgrowth, chemical inhibition of auxin synthesis phenocopies cytokinin application. Finally, cytokinin-insensitive mutants resemble plants with altered auxin signaling and are hypersensitive to auxin. In summary, our results suggest that the ratio between auxin and cytokinin signaling is the basis for developmental decisions in the moss gametophore.

DRMY1 promotes robust morphogenesis by sustaining translation of a hormone signaling protein.

Robustness is the invariant development of phenotype despite environmental changes and genetic perturbations. In the Arabidopsis flower bud, four sepals initiate at robust positions and times and grow to equal size to enclose and protect the inner floral organs. We previously characterized the mutant development related myb-like1 (drmy1), where 3-5 sepals initiate at irregular positions and variable times and grow to different sizes, compromising their protective function. The molecular mechanism underlying this loss of robustness was unclear. Here, we show that drmy1 has reduced TARGET OF RAPAMYCIN (TOR) activity, ribosomal content, and translation. Translation reduction decreases the protein level of ARABIDOPSIS RESPONSE REGULATOR7 (ARR7), a rapidly synthesized and degraded cytokinin signaling inhibitor. The resultant upregulation of cytokinin signaling disrupts the robust positioning of auxin signaling, causing variable sepal initiation. Our work shows that the homeostasis of translation, a ubiquitous cellular process, is crucial for the robust spatiotemporal patterning of organogenesis.

An optimized pipeline for live imaging whole Arabidopsis leaves at cellular resolution.

BACKGROUND: Live imaging is the gold standard for determining how cells give rise to organs. However, tracking many cells across whole organs over large developmental time windows is extremely challenging. In this work, we provide a comparably simple method for confocal live imaging entire Arabidopsis thaliana first leaves across early development. Our imaging method works for both wild-type leaves and the complex curved leaves of the jaw-1D mutant. RESULTS: We find that dissecting the cotyledons, affixing a coverslip above the samples and mounting samples with perfluorodecalin yields optimal imaging series for robust cellular and organ level analysis. We provide details of our complementary image processing steps in MorphoGraphX software for segmenting, tracking lineages, and measuring a suite of cellular properties. We also provide MorphoGraphX image processing scripts we developed to automate analysis of segmented images and data presentation. CONCLUSIONS: Our imaging techniques and processing steps combine into a robust imaging pipeline. With this pipeline we are able to examine important nuances in the cellular growth and differentiation of jaw-D versus WT leaves that have not been demonstrated before. Our pipeline is approachable and easy to use for leaf development live imaging.

Enhancer activation via TCP and HD-ZIP and repression by Dof transcription factors mediate giant cell-specific expression.

Proper cell-type identity relies on highly coordinated regulation of gene expression. Regulatory elements such as enhancers can produce cell type-specific expression patterns, but the mechanisms underlying specificity are not well understood. We previously identified an enhancer region capable of driving specific expression in giant cells, which are large, highly endoreduplicated cells in the Arabidopsis thaliana sepal epidermis. In this study, we use the giant cell enhancer as a model to understand the regulatory logic that promotes cell type-specific expression. Our dissection of the enhancer revealed that giant cell specificity is mediated primarily through the combination of two activators and one repressor. HD-ZIP and TCP transcription factors are involved in the activation of expression throughout the epidermis. High expression of HD-ZIP transcription factor genes in giant cells promoted higher expression driven by the enhancer in giant cells. Dof transcription factors repressed the activity of the enhancer such that only giant cells maintained enhancer activity. Thus, our data are consistent with a conceptual model whereby cell type-specific expression emerges from the combined activities of three transcription factor families activating and repressing expression in epidermal cells.

Cytokinin-CLAVATA cross-talk is an ancient mechanism regulating shoot meristem homeostasis in land plants.

SignificancePlants grow from their tips. The gametophore (shoot-like organ) tip of the moss Physcomitrium patens is a single cell that performs the same functions as those of multicellular flowering plants, producing the cells that make leaves and regenerating new stem cells to maintain the shoot tip. Several pathways, including CLAVATA and cytokinin hormonal signaling, regulate stem cell abundance in flowering plants and in mosses, although the mechanisms whereby these pathways regulate stem cell abundance and their conservation between these plant lineages is poorly understood. Using moss, we investigated how PpCLAVATA and cytokinin signaling interact. Overall, we found evidence that PpCLAVATA and cytokinin signaling interact similarly in moss and flowering plants, despite their distinct anatomies, life cycles, and evolutionary distance.

Stepping on the molecular brake: Slowing down proliferation to allow differentiation.

Cellular differentiation can entail changes to the cell cycle. In this issue of Developmental Cell, Han et al. show that the transcription factor MUTE directly activates expression of the cyclin-dependent kinase (CDK) inhibitor SIAMESE RELATED 4 (SMR4), thereby slowing down G1 during the transition to stomatal differentiation.

Fifteen compelling open questions in plant cell biology.

As scientists, we are at least as excited about the open questions-the things we do not know-as the discoveries. Here, we asked 15 experts to describe the most compelling open questions in plant cell biology. These are their questions: How are organelle identity, domains, and boundaries maintained under the continuous flux of vesicle trafficking and membrane remodeling? Is the plant cortical microtubule cytoskeleton a mechanosensory apparatus? How are the cellular pathways of cell wall synthesis, assembly, modification, and integrity sensing linked in plants? Why do plasmodesmata open and close? Is there retrograde signaling from vacuoles to the nucleus? How do root cells accommodate fungal endosymbionts? What is the role of cell edges in plant morphogenesis? How is the cell division site determined? What are the emergent effects of polyploidy on the biology of the cell, and how are any such "rules" conditioned by cell type? Can mechanical forces trigger new cell fates in plants? How does a single differentiated somatic cell reprogram and gain pluripotency? How does polarity develop de-novo in isolated plant cells? What is the spectrum of cellular functions for membraneless organelles and intrinsically disordered proteins? How do plants deal with internal noise? How does order emerge in cells and propagate to organs and organisms from complex dynamical processes? We hope you find the discussions of these questions thought provoking and inspiring.

A Life Cycle for Modeling Biology at Different Scales.

Modeling has become a popular tool for inquiry and discovery across biological disciplines. Models allow biologists to probe complex questions and to guide experimentation. Modeling literacy among biologists, however, has not always kept pace with the rise in popularity of these techniques and the relevant advances in modeling theory. The result is a lack of understanding that inhibits communication and ultimately, progress in data gathering and analysis. In an effort to help bridge this gap, we present a blueprint that will empower biologists to interrogate and apply models in their field. We demonstrate the applicability of this blueprint in two case studies from distinct subdisciplines of biology; developmental-biomechanics and evolutionary biology. The models used in these fields vary from summarizing dynamical mechanisms to making statistical inferences, demonstrating the breadth of the utility of models to explore biological phenomena.