Mechanical forces in plant tissue matrix orient cell divisions via microtubule stabilization.

Plant morphogenesis relies exclusively on oriented cell expansion and division. Nonetheless, the mechanism(s) determining division plane orientation remain elusive. Here, we studied tissue healing after laser-assisted wounding in roots of Arabidopsis thaliana and uncovered how mechanical forces stabilize and reorient the microtubule cytoskeleton for the orientation of cell division. We identified that root tissue functions as an interconnected cell matrix, with a radial gradient of tissue extendibility causing predictable tissue deformation after wounding. This deformation causes instant redirection of expansion in the surrounding cells and reorientation of microtubule arrays, ultimately predicting cell division orientation. Microtubules are destabilized under low tension, whereas stretching of cells, either through wounding or external aspiration, immediately induces their polymerization. The higher microtubule abundance in the stretched cell parts leads to the reorientation of microtubule arrays and, ultimately, informs cell division planes. This provides a long-sought mechanism for flexible re-arrangement of cell divisions by mechanical forces for tissue reconstruction and plant architecture.

Growth and tension in explosive fruit.

Exploding seed pods of the common weed Cardamine hirsuta have the remarkable ability to launch seeds far from the plant. The energy for this explosion comes from tension that builds up in the fruit valves. Above a critical threshold, the fruit fractures along its dehiscence zone and the two valves coil explosively, ejecting the seeds. A common mechanism to generate tension is drying, causing tissues to shrink. However, this does not happen in C. hirsuta fruit. Instead, tension is produced by active contraction of growing exocarp cells in the outer layer of the fruit valves. Exactly how growth causes the exocarp tissue to contract and generate pulling force is unknown. Here we show that the reorientation of microtubules in the exocarp cell cortex changes the orientation of cellulose microfibrils in the cell wall and the consequent cellular growth pattern. We used mechanical modeling to show how tension emerges through growth due to the highly anisotropic orientation of load-bearing cellulose microfibrils and their effect on cell shape. By explicitly defining the cell wall as multi-layered in our model, we discovered that a cross-lamellate pattern of cellulose microfibrils further enhances the developing tension in growing cells. Therefore, the interplay of cell wall properties with turgor-driven growth enables the fruit exocarp to generate sufficient tension to power explosive seed dispersal.

Quantitative analysis of 3D cellular geometry and modelling of the Arabidopsis embryo.

As many multicellular organisms, land plants start their life as a single cell, which forms an embryo. Embryo morphology is relatively simple, yet comprises basic tissues and organs, as well as stem cells that sustain post-embryonic development. Being condensed in both time and space, early plant embryogenesis offers an excellent window to study general principles of plant development. However, it has been technically challenging to obtain high spatial microscopic resolution, or to perform live imaging, that would enable an in-depth investigation. Recent advances in sample preparation and microscopy now allow studying the detailed cellular morphology of plant embryos in 3D. When coupled to quantitative image analysis and computational modelling, this allows resolving the temporal and spatial interactions between cellular patterning and genetic networks. In this review, we discuss examples of interdisciplinary studies that showcase the potential of the early plant embryo for revealing principles underlying plant development.

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.

Cytokinin promotes growth cessation in the Arabidopsis root.

The Arabidopsis root offers good opportunities to investigate how regulated cellular growth shapes different tissues and organs, a key question in developmental biology. Along the root's longitudinal axis, cells sequentially occupy different developmental states. Proliferative meristematic cells give rise to differentiating cells, which rapidly elongate in the elongation zone, then mature and stop growing in the differentiation zone. The phytohormone cytokinin contributes to this zonation by positioning the boundary between the meristem and the elongation zone, called the transition zone. However, the cellular growth profile underlying root zonation is not well understood, and the cellular mechanisms that mediate growth cessation remain unclear. By using time-lapse imaging, genetics, and computational analysis, we analyze the effect of cytokinin on root zonation and cellular growth. We found that cytokinin promotes growth cessation in the distal (shootward) elongation zone in conjunction with accelerating the transition from elongation to differentiation. We estimated cell-wall stiffness by using osmotic treatment experiments and found that cytokinin-mediated growth cessation is associated with cell-wall stiffening and requires the action of an auxin influx carrier, AUX1. Our measurement of growth and cell-wall mechanical properties at a cellular resolution reveal mechanisms via which cytokinin influences cell behavior to shape tissue patterns.

Wounding-induced changes in cellular pressure and localized auxin signalling spatially coordinate restorative divisions in roots.

Wound healing in plant tissues, consisting of rigid cell wall-encapsulated cells, represents a considerable challenge and occurs through largely unknown mechanisms distinct from those in animals. Owing to their inability to migrate, plant cells rely on targeted cell division and expansion to regenerate wounds. Strict coordination of these wound-induced responses is essential to ensure efficient, spatially restricted wound healing. Single-cell tracking by live imaging allowed us to gain mechanistic insight into the wound perception and coordination of wound responses after laser-based wounding in Arabidopsis root. We revealed a crucial contribution of the collapse of damaged cells in wound perception and detected an auxin increase specific to cells immediately adjacent to the wound. This localized auxin increase balances wound-induced cell expansion and restorative division rates in a dose-dependent manner, leading to tumorous overproliferation when the canonical TIR1 auxin signaling is disrupted. Auxin and wound-induced turgor pressure changes together also spatially define the activation of key components of regeneration, such as the transcription regulator ERF115. Our observations suggest that the wound signaling involves the sensing of collapse of damaged cells and a local auxin signaling activation to coordinate the downstream transcriptional responses in the immediate wound vicinity.

Re-activation of Stem Cell Pathways for Pattern Restoration in Plant Wound Healing.

Patterning in plants relies on oriented cell divisions and acquisition of specific cell identities. Plants regularly endure wounds caused by abiotic or biotic environmental stimuli and have developed extraordinary abilities to restore their tissues after injuries. Here, we provide insight into a mechanism of restorative patterning that repairs tissues after wounding. Laser-assisted elimination of different cells in Arabidopsis root combined with live-imaging tracking during vertical growth allowed analysis of the regeneration processes in vivo. Specifically, the cells adjacent to the inner side of the injury re-activated their stem cell transcriptional programs. They accelerated their progression through cell cycle, coordinately changed the cell division orientation, and ultimately acquired de novo the correct cell fates to replace missing cells. These observations highlight existence of unknown intercellular positional signaling and demonstrate the capability of specified cells to re-acquire stem cell programs as a crucial part of the plant-specific mechanism of wound healing.

A SOSEKI-based coordinate system interprets global polarity cues in Arabidopsis.

Multicellular development requires coordinated cell polarization relative to body axes, and translation to oriented cell division1-3. In plants, it is unknown how cell polarities are connected to organismal axes and translated to division. Here, we identify Arabidopsis SOSEKI proteins that integrate apical-basal and radial organismal axes to localize to polar cell edges. Localization does not depend on tissue context, requires cell wall integrity and is defined by a transferrable, protein-specific motif. A Domain of Unknown Function in SOSEKI proteins resembles the DIX oligomerization domain in the animal Dishevelled polarity regulator. The DIX-like domain self-interacts and is required for edge localization and for influencing division orientation, together with a second domain that defines the polar membrane domain. Our work shows that SOSEKI proteins locally interpret global polarity cues and can influence cell division orientation. Furthermore, this work reveals that, despite fundamental differences, cell polarity mechanisms in plants and animals converge on a similar protein domain.

Auxin response cell-autonomously controls ground tissue initiation in the early Arabidopsis embryo.

Plant organs are typically organized into three main tissue layers. The middle ground tissue layer comprises the majority of the plant body and serves a wide range of functions, including photosynthesis, selective nutrient uptake and storage, and gravity sensing. Ground tissue patterning and maintenance in Arabidopsis are controlled by a well-established gene network revolving around the key regulator SHORT-ROOT (SHR). In contrast, it is completely unknown how ground tissue identity is first specified from totipotent precursor cells in the embryo. The plant signaling molecule auxin, acting through AUXIN RESPONSE FACTOR (ARF) transcription factors, is critical for embryo patterning. The auxin effector ARF5/MONOPTEROS (MP) acts both cell-autonomously and noncell-autonomously to control embryonic vascular tissue formation and root initiation, respectively. Here we show that auxin response and ARF activity cell-autonomously control the asymmetric division of the first ground tissue cells. By identifying embryonic target genes, we show that MP transcriptionally initiates the ground tissue lineage and acts upstream of the regulatory network that controls ground tissue patterning and maintenance. Strikingly, whereas the SHR network depends on MP, this MP function is, at least in part, SHR independent. Our study therefore identifies auxin response as a regulator of ground tissue specification in the embryonic root, and reveals that ground tissue initiation and maintenance use different regulators and mechanisms. Moreover, our data provide a framework for the simultaneous formation of multiple cell types by the same transcriptional regulator.

Correction: Centering the Organizing Center in the Arabidopsis thaliana Shoot Apical Meristem by a Combination of Cytokinin Signaling and Self-Organization.

[This corrects the article DOI: 10.1371/journal.pone.0147830.].

Centering the Organizing Center in the Arabidopsis thaliana Shoot Apical Meristem by a Combination of Cytokinin Signaling and Self-Organization.

Plants have the ability to continously generate new organs by maintaining populations of stem cells throught their lives. The shoot apical meristem (SAM) provides a stable environment for the maintenance of stem cells. All cells inside the SAM divide, yet boundaries and patterns are maintained. Experimental evidence indicates that patterning is independent of cell lineage, thus a dynamic self-regulatory mechanism is required. A pivotal role in the organization of the SAM is played by the WUSCHEL gene (WUS). An important question in this regard is that how WUS expression is positioned in the SAM via a cell-lineage independent signaling mechanism. In this study we demonstrate via mathematical modeling that a combination of an inhibitor of the Cytokinin (CK) receptor, Arabidopsis histidine kinase 4 (AHK4) and two morphogens originating from the top cell layer, can plausibly account for the cell lineage-independent centering of WUS expression within SAM. Furthermore, our laser ablation and microsurgical experiments support the hypothesis that patterning in SAM occurs at the level of CK reception and signaling. The model suggests that the interplay between CK signaling, WUS/CLV feedback loop and boundary signals can account for positioning of the WUS expression, and provides directions for further experimental investigation.

Reporters for sensitive and quantitative measurement of auxin response.

The visualization of hormonal signaling input and output is key to understanding how multicellular development is regulated. The plant signaling molecule auxin triggers many growth and developmental responses, but current tools lack the sensitivity or precision to visualize these. We developed a set of fluorescent reporters that allow sensitive and semiquantitative readout of auxin responses at cellular resolution in Arabidopsis thaliana. These generic tools are suitable for any transformable plant species.

Plant development. Integration of growth and patterning during vascular tissue formation in Arabidopsis.

Coordination of cell division and pattern formation is central to tissue and organ development, particularly in plants where walls prevent cell migration. Auxin and cytokinin are both critical for division and patterning, but it is unknown how these hormones converge upon tissue development. We identify a genetic network that reinforces an early embryonic bias in auxin distribution to create a local, nonresponding cytokinin source within the root vascular tissue. Experimental and theoretical evidence shows that these cells act as a tissue organizer by positioning the domain of oriented cell divisions. We further demonstrate that the auxin-cytokinin interaction acts as a spatial incoherent feed-forward loop, which is essential to generate distinct hormonal response zones, thus establishing a stable pattern within a growing vascular tissue.

Genetic control of plant development by overriding a geometric division rule.

Formative cell divisions are critical for multicellular patterning. In the early plant embryo, such divisions follow from orienting the division plane. A major unanswered question is how division plane orientation is genetically controlled, and in particular whether this relates to cell geometry. We have generated a complete 4D map of early Arabidopsis embryogenesis and used computational analysis to demonstrate that several divisions follow a rule that uses the smallest wall area going through the center of the cell. In other cases, however, cell division clearly deviates from this rule, which invariably leads to asymmetric cell division. By analyzing mutant embryos and through targeted genetic perturbation, we show that response to the hormone auxin triggers a deviation from the "shortest wall" rule. Our work demonstrates that a simple default rule couples division orientation to cell geometry in the embryo and that genetic regulation can create patterns by overriding the default rule.

A bHLH complex controls embryonic vascular tissue establishment and indeterminate growth in Arabidopsis.

Plants have a remarkable potential for sustained (indeterminate) postembryonic growth. Following their specification in the early embryo, tissue-specific precursor cells first establish tissues and later maintain them postembryonically. The mechanisms underlying these processes are largely unknown. Here we define local control of oriented, periclinal cell division as the mechanism underlying both the establishment and maintenance of vascular tissue. We identify an auxin-regulated basic helix-loop-helix (bHLH) transcription factor dimer as a critical regulator of vascular development. Due to a loss of periclinal divisions, vascular tissue gradually disappears in bHLH-deficient mutants; conversely, ectopic expression is sufficient for triggering periclinal divisions. We show that this dimer operates independently of tissue identity but is restricted to a small vascular domain by integrating overlapping transcription patterns of the interacting bHLH proteins. Our work reveals a common mechanism for tissue establishment and indeterminate vascular development and provides a conceptual framework for developmental control of local cell divisions.

Auxin regulation of embryonic root formation.

The plant hormone auxin was initially identified as the bioactive substance that induces roots in plant tissue culture. In the past decades, mechanisms for auxin action, including its transport and response, have been described in detail. However, a molecular and cellular description of its role in root initiation is far from complete. In this review, we discuss recent advances in our understanding of auxin-dependent embryonic root formation. During this process, a root meristem is initiated in a precise and predictable position, and at a stage when the organism consists of relatively few cells. Recent studies have revealed mechanisms for local control of auxin transport, for cellular differences in auxin response components and cell type-specific chromatin regulation. The recent identification of biologically relevant target genes for auxin regulation during embryonic root initiation now also allows dissection of auxin-activated cellular processes. Finally, we discuss the potential for hormonal cross-regulation in embryonic root formation.

Stem cell activation by light guides plant organogenesis.

Leaves originate from stem cells located at the shoot apical meristem. The meristem is shielded from the environment by older leaves, and leaf initiation is considered to be an autonomous process that does not depend on environmental cues. Here we show that light acts as a morphogenic signal that controls leaf initiation and stabilizes leaf positioning. Leaf initiation in tomato shoot apices ceases in the dark but resumes in the light, an effect that is mediated through the plant hormone cytokinin. Dark treatment also affects the subcellular localization of the auxin transporter PIN1 and the concomitant formation of auxin maxima. We propose that cytokinin is required for meristem propagation, and that auxin redirects cytokinin-inducible meristem growth toward organ formation. In contrast to common wisdom over the last 150 years, the light environment controls the initiation of lateral organs by regulating two key hormones: auxin and cytokinin.

Comprehensive analysis of the regulatory roles of auxin in early transdifferentiation into xylem cells.

Auxin is essential for the formation of the vascular system. We previously reported that a polar auxin transport inhibitor, 1-N-naphthylphthalamic acid (NPA) decreased intracellular auxin levels and prevented tracheary element (TE) differentiation from isolated Zinnia mesophyll cells, but that additional auxin, 1-naphthaleneacetic acid (NAA) overcame this inhibition. To understand the role of auxin in gene regulation during TE differentiation, we performed microarray analysis of genes expressed in NPA-treated cells and NPA-NAA-treated cells. The systematic gene expression analysis revealed that NAA promoted the expression of genes related to auxin signaling and transcription factors that are known to be key regulators of differentiation of procambial and xylem precursor cells. NAA also promoted the expression of genes related to biosynthesis and metabolism of other plant hormones, such as cytokinin, gibberellin and brassinosteroid. Interestingly, detailed analysis showed that NAA rapidly induces the expression of auxin carrier gene homologues. It suggested a positive feedback loop for auxin-regulating vascular differentiation. Based on these results, we discuss the auxin function in early processes of transdifferentiation into TEs.

Inhibition of transdifferentiation into tracheary elements by polar auxin transport inhibitors through intracellular auxin depletion.

Polar auxin transport is essential for the formation of continuous vascular strands in the plant body. To understand its mechanism, polar auxin transport inhibitors have often been used. However, the role of auxin in vascular differentiation at the unicellular level has remained elusive. Using a Zinnia elegans cell culture system, in which single mesophyll cells transdifferentiate into tracheary elements (TEs), we demonstrated that auxin transport inhibitors prevented TE differentiation and that high concentrations of 1-naphthaleneacetic acid (NAA) and IAA overcame the repression of TE differentiation. Measurements of NAA accumulation with 3H-labeled NAA in the presence or absence of 1-N-naphthylphthalamic acid (NPA) revealed enhanced NAA accumulation within the cell. In the NPA-treated cells, intracellular free NAA decreased, while its metabolites increased. Therefore, the polar auxin transport inhibitors may prevent auxin efflux and consequently promote NAA accumulation in Zinnia cells. The excess intracellular NAA may also activate NAA metabolism, resulting in a decrease in free NAA levels. This depletion of free NAA may prevent TE differentiation. The decreased auxin activity in NPA-treated cells was confirmed by the fact that the DR5 (a synthetic auxin-inducible promoter)-mediated expression of a reporter protein was suppressed in such cells. Gene expression analysis indicated that NPA suppressed TE differentiation at an early process of transdifferentiation into TEs. Based on these results, the inter-relationship between auxin and vascular cell development at a cellular level is discussed.