Multi-scale mechanisms driving root regeneration: From regeneration competence to tissue repatterning.

Plants possess an outstanding capacity to regenerate enabling them to repair damages caused by suboptimal environmental conditions, biotic attacks, or mechanical damages impacting the survival of these sessile organisms. Although the extent of regeneration varies greatly between localized cell damage and whole organ recovery, the process of regeneration can be subdivided into a similar sequence of interlinked regulatory processes. That is, competence to regenerate, cell fate reprogramming, and the repatterning of the tissue. Here, using root tip regeneration as a paradigm system to study plant regeneration, we provide a synthesis of the molecular responses that underlie both regeneration competence and the repatterning of the root stump. Regarding regeneration competence, we discuss the role of wound signaling, hormone responses and synthesis, and rapid changes in gene expression observed in the cells close to the cut. Then, we consider how this rapid response is followed by the tissue repatterning phase, where cells experience cell fate changes in a spatial and temporal order to recreate the lost stem cell niche and columella. Lastly, we argue that a multi-scale modeling approach is fundamental to uncovering the mechanisms underlying root regeneration, as it allows to integrate knowledge of cell-level gene expression, cell-to-cell transport of hormones and transcription factors, and tissue-level growth dynamics to reveal how the bi-directional feedbacks between these processes enable self-organized repatterning of the root apex.

Complementary roles for auxin and auxin signalling revealed by reverse engineering lateral root stable prebranch site formation.

Priming is the process through which periodic elevations in auxin signalling prepattern future sites for lateral root formation, called prebranch sites. Thus far, the extent to which elevations in auxin concentration and/or auxin signalling are required for priming and prebranch site formation has remained a matter of debate. Recently, we discovered a reflux-and-growth mechanism for priming generating periodic elevations in auxin concentration that subsequently dissipate. Here, we reverse engineer a mechanism for prebranch site formation that translates these transient elevations into a persistent increase in auxin signalling, resolving the prior debate into a two-step process of auxin concentration-mediated initial signal and auxin signalling capacity-mediated memorization. A crucial aspect of the prebranch site formation mechanism is its activation in response to time-integrated rather than instantaneous auxin signalling. The proposed mechanism is demonstrated to be consistent with prebranch site auxin signalling dynamics, lateral inhibition, and symmetry-breaking mechanisms and perturbations in auxin homeostasis.

GH3-mediated auxin inactivation attenuates multiple stages of lateral root development.

Lateral root (LR) positioning and development rely on the dynamic interplay between auxin production, transport but also inactivation. Nonetheless, how the latter affects LR organogenesis remains largely uninvestigated. Here, we systematically analyze the impact of the major auxin inactivation pathway defined by GRETCHEN HAGEN3-type (GH3) auxin conjugating enzymes and DIOXYGENASE FOR AUXIN OXIDATION1 (DAO1) in all stages of LR development using reporters, genetics and inhibitors in Arabidopsis thaliana. Our data demonstrate that the gh3.1/2/3/4/5/6 hextuple (gh3hex) mutants display a higher LR density due to increased LR initiation and faster LR developmental progression, acting epistatically over dao1-1. Grafting and local inhibitor applications reveal that root and shoot GH3 activities control LR formation. The faster LR development in gh3hex is associated with GH3 expression domains in and around developing LRs. The increase in LR initiation is associated with accelerated auxin response oscillations coinciding with increases in apical meristem size and LR cap cell death rates. Our research reveals how GH3-mediated auxin inactivation attenuates LR development. Local GH3 expression in LR primordia attenuates development and emergence, whereas GH3 effects on pre-initiation stages are indirect, by modulating meristem activities that in turn coordinate root growth with LR spacing.

Of mice and plants: Comparative developmental systems biology.

Multicellular animals and plants represent independent evolutionary experiments with complex multicellular bodyplans. Differences in their life history, a mobile versus sessile lifestyle, and predominant embryonic versus postembryonic development, have led to the evolution of highly different body plans. However, also many intriguing parallels exist. Extension of the vertebrate body axis and its segmentation into somites bears striking resemblance to plant root growth and the concomittant prepatterning of lateral root competent sites. Likewise, plant shoot phyllotaxis displays similarities with vertebrate limb and digit patterning. Additionally, both plants and animals use complex signalling systems combining systemic and local signals to fine tune and coordinate organ growth across their body. Identification of these striking examples of convergent evolution provides support for the existence of general design principles: the idea that for particular patterning demands, evolution is likely to arrive at highly similar developmental patterning mechanisms. Furthermore, focussing on these parallels may aid in identifying core mechanistic principles, often obscured by the highly complex nature of multiscale patterning processes.

Modelling the physiological relevance of sucrose export repression by an Flowering Time homolog in the long-distance phloem of potato.

Yield of harvestable plant organs depends on photosynthetic assimilate production in source leaves, long-distance sucrose transport and sink-strength. While photosynthesis optimization has received considerable interest for optimizing plant yield, the potential for improving long-distance sucrose transport has received far less attention. Interestingly, a recent potato study demonstrates that the tuberigen StSP6A binds to and reduces activity of the StSWEET11 sucrose exporter. While the study suggested that reducing phloem sucrose efflux may enhance tuber yield, the precise mechanism and physiological relevance of this effect remained an open question. Here, we develop the first mechanistic model for sucrose transport, parameterized for potato plants. The model incorporates SWEET-mediated sucrose export, SUT-mediated sucrose retrieval from the apoplast and StSP6A-StSWEET11 interactions. Using this model, we were able to substantiate the physiological relevance of the StSP6A-StSWEET11 interaction in the long-distance phloem for potato tuber yield, as well as to show the non-linear nature of this effect.

Bootstrapping and Pinning down the Root Meristem; the Auxin-PLT-ARR Network Unites Robustness and Sensitivity in Meristem Growth Control.

After germination, the meristem of the embryonic plant root becomes activated, expands in size and subsequently stabilizes to support post-embryonic root growth. The plant hormones auxin and cytokinin, together with master transcription factors of the PLETHORA (PLT) family have been shown to form a regulatory network that governs the patterning of this root meristem. Still, which functional constraints contributed to shaping the dynamics and architecture of this network, has largely remained unanswered. Using a combination of modeling approaches we reveal how the interplay between auxin and PLTs enables meristem activation in response to above-threshold stimulation, while its embedding in a PIN-mediated auxin reflux loop ensures localized PLT transcription and thereby, a finite meristem size. We furthermore demonstrate how this constrained PLT transcriptional domain enables independent control of meristem size and division rates, further supporting a division of labor between auxin and PLT. We subsequently reveal how the weaker auxin antagonism of the earlier active Arabidopsis response regulator 12 (ARR12) may arise from the absence of a DELLA protein interaction domain. Our model indicates that this reduced strength is essential to prevent collapse in the early stages of meristem expansion while at later stages the enhanced strength of Arabidopsis response regulator 1 (ARR1) is required for sufficient meristem size control. Summarizing, our work indicates that functional constraints significantly contribute to shaping the auxin-cytokinin-PLT regulatory network.

Halotropism requires phospholipase Dζ1-mediated modulation of cellular polarity of auxin transport carriers.

Endocytosis and relocalization of auxin carriers represent important mechanisms for adaptive plant growth and developmental responses. Both root gravitropism and halotropism have been shown to be dependent on relocalization of auxin transporters. Following their homology to mammalian phospholipase Ds (PLDs), plant PLDζ-type enzymes are likely candidates to regulate auxin carrier endocytosis. We investigated root tropic responses for an Arabidopsis pldζ1-KO mutant and its effect on the dynamics of two auxin transporters during salt stress, that is, PIN2 and AUX1. We found altered root growth and halotropic and gravitropic responses in the absence of PLDζ1 and report a role for PLDζ1 in the polar localization of PIN2. Additionally, irrespective of the genetic background, salt stress induced changes in AUX1 polarity. Utilizing our previous computational model, we found that these novel salt-induced AUX1 changes contribute to halotropic auxin asymmetry. We also report the formation of "osmotic stress-induced membrane structures." These large membrane structures are formed at the plasma membrane shortly after NaCl or sorbitol treatment and have a prolonged presence in a pldζ1 mutant. Taken together, these results show a crucial role for PLDζ1 in both ionic and osmotic stress-induced auxin carrier dynamics during salt stress.

Periodic Lateral Root Priming: What Makes It Tick?

Conditioning small groups of root pericycle cells for future lateral root formation has a major impact on overall plant root architecture. This priming of lateral roots occurs rhythmically, involving temporal oscillations in auxin response in the root tip. During growth, this process generates a spatial pattern of prebranch sites, an early stage in lateral root formation characterized by a stably maintained high auxin response. To date, the molecular mechanism behind this rhythmicity has remained elusive. Some data implicate a cell-autonomous oscillation in gene expression, while others strongly support the importance of tissue-level modulations in auxin fluxes. Here, we summarize the experimental data on periodic lateral root priming. We present a theoretical framework that distinguishes between a priming signal and its subsequent memorization and show how major roles for auxin fluxes and gene expression naturally emerge from this framework. We then discuss three mechanisms that could potentially induce oscillations of auxin response: cell-autonomous oscillations, Turing-type patterning, and tissue-level oscillations in auxin fluxes, along with specific properties of lateral root priming that may be used to discern which type of mechanism is most likely to drive lateral root patterning. We conclude with suggestions for future experiments and modeling studies.

PLETHORA gradient formation mechanism separates auxin responses.

During plant growth, dividing cells in meristems must coordinate transitions from division to expansion and differentiation, thus generating three distinct developmental zones: the meristem, elongation zone and differentiation zone. Simultaneously, plants display tropisms, rapid adjustments of their direction of growth to adapt to environmental conditions. It is unclear how stable zonation is maintained during transient adjustments in growth direction. In Arabidopsis roots, many aspects of zonation are controlled by the phytohormone auxin and auxin-induced PLETHORA (PLT) transcription factors, both of which display a graded distribution with a maximum near the root tip. In addition, auxin is also pivotal for tropic responses. Here, using an iterative experimental and computational approach, we show how an interplay between auxin and PLTs controls zonation and gravitropism. We find that the PLT gradient is not a direct, proportionate readout of the auxin gradient. Rather, prolonged high auxin levels generate a narrow PLT transcription domain from which a gradient of PLT protein is subsequently generated through slow growth dilution and cell-to-cell movement. The resulting PLT levels define the location of developmental zones. In addition to slowly promoting PLT transcription, auxin also rapidly influences division, expansion and differentiation rates. We demonstrate how this specific regulatory design in which auxin cooperates with PLTs through different mechanisms and on different timescales enables both the fast tropic environmental responses and stable zonation dynamics necessary for coordinated cell differentiation.

Modeling halotropism: a key role for root tip architecture and reflux loop remodeling in redistributing auxin.

A key characteristic of plant development is its plasticity in response to various and dynamically changing environmental conditions. Tropisms contribute to this flexibility by allowing plant organs to grow from or towards environmental cues. Halotropism is a recently described tropism in which plant roots bend away from salt. During halotropism, as in most other tropisms, directional growth is generated through an asymmetric auxin distribution that generates differences in growth rate and hence induces bending. Here, we develop a detailed model of auxin transport in the Arabidopsis root tip and combine this with experiments to investigate the processes generating auxin asymmetry during halotropism. Our model points to the key role of root tip architecture in allowing the decrease in PIN2 at the salt-exposed side of the root to result in a re-routing of auxin to the opposite side. In addition, our model demonstrates how feedback of auxin on the auxin transporter AUX1 amplifies this auxin asymmetry, while a salt-induced transient increase in PIN1 levels increases the speed at which this occurs. Using AUX1-GFP imaging and pin1 mutants, we experimentally confirmed these model predictions, thus expanding our knowledge of the cellular basis of halotropism.

Modelling asymmetric somitogenesis: Deciphering the mechanisms behind species differences.

Somitogenesis is one of the major hallmarks of bilateral symmetry in vertebrates. This symmetry is lost when retinoic acid (RA) signalling is inhibited, allowing the left-right determination pathway to influence somitogenesis. In all three studied vertebrate model species, zebrafish, chicken and mouse, the frequency of somite formation becomes asymmetric, with slower gene expression oscillations driving somitogenesis on the right side. Still, intriguingly, the resulting left-right asymmetric phenotypes differ significantly between these model species. While somitogenesis is generally considered as functionally equivalent among different vertebrates, substantial differences exist in the subset of oscillating genes between different vertebrate species. Variation also appears to exist in the way oscillations cease and somite boundaries become patterned. In addition, in absence of RA, the FGF8 gradient thought to constitute the determination wavefront becomes asymmetric in zebrafish and mouse, extending more anteriorly to the right, while remaining symmetric in chicken. Here we use a computational modelling approach to decipher the causes underlying species differences in asymmetric somitogenesis. Specifically, we investigate to what extent differences can be explained from observed differences in FGF asymmetry and whether differences in somite determination dynamics may also be involved. We demonstrate that a simple clock-and-wavefront model incorporating the observed left-right differences in somitogenesis frequency readily reproduces asymmetric somitogenesis in chicken. However, incorporating asymmetry in FGF signalling was insufficient to robustly reproduce mouse or zebrafish asymmetry phenotypes. In order to explain these phenoptypes we needed to extend the basic model, incorporating species-specific details of the somitogenesis determination mechanism. Our results thus demonstrate that a combination of differences in FGF dynamics and somite determination cause species differences in asymmetric somitogenesis. In addition,they highlight the power of using computational models as well as studying left-right asymmetry to obtain more insight in somitogenesis.

Auxin Information Processing; Partners and Interactions beyond the Usual Suspects.

Auxin plays a major role in a variety of processes involved in plant developmental patterning and its adaptation to environmental conditions. Therefore, an important question is how specificity in auxin signalling is achieved, that is, how a single signalling molecule can carry so many different types of information. In recent years, many studies on auxin specificity have been published, unravelling increasingly more details on differential auxin sensitivity, expression domains and downstream partners of the auxin receptors (transport inhibitor response 1 (TIR1) and other auxin signaling F-box proteins (AFB)), transcriptional repressors that are degraded in response to auxin (AUX/IAA) and downstream auxin response factors (ARF) that together constitute the plant's major auxin response pathways. These data are critical to explain how, in the same cells, different auxin levels may trigger different responses, as well as how in different spatial or temporal contexts similar auxin signals converge to different responses. However, these insights do not yet answer more complex questions regarding auxin specificity. As an example, they leave open the question of how similar sized auxin changes at similar locations result in different responses depending on the duration and spatial extent of the fluctuation in auxin levels. Similarly, it leaves unanswered how, in the case of certain tropisms, small differences in signal strength at both sides of a plant organ are converted into an instructive auxin asymmetry that enables a robust tropic response. Finally, it does not explain how, in certain cases, substantially different auxin levels become translated into similar cellular responses, while in other cases similar auxin levels, even when combined with similar auxin response machinery, may trigger different responses. In this review, we illustrate how considering the regulatory networks and contexts in which auxin signalling takes place helps answer these types of fundamental questions.

Polar auxin transport: models and mechanisms.

Spatial patterns of the hormone auxin are important drivers of plant development. The observed feedback between the active, directed transport that generates auxin patterns and the auxin distribution that influences transport orientation has rendered this a popular subject for modelling studies. Here we propose a new mathematical framework for the analysis of polar auxin transport and present a detailed mathematical analysis of published models. We show that most models allow for self-organised patterning for similar biological assumptions, and find that the pattern generated is typically unidirectional, unless additional assumptions or mechanisms are incorporated. Our analysis thus suggests that current models cannot explain the bidirectional fountain-type patterns found in plant meristems in a fully self-organised manner, and we discuss future research directions to address the gaps in our understanding of auxin transport mechanisms.

Segment-specific adhesion as a driver of convergent extension.

Convergent extension, the simultaneous extension and narrowing of tissues, is a crucial event in the formation of the main body axis during embryonic development. It involves processes on multiple scales: the sub-cellular, cellular and tissue level, which interact via explicit or intrinsic feedback mechanisms. Computational modelling studies play an important role in unravelling the multiscale feedbacks underlying convergent extension. Convergent extension usually operates in tissue which has been patterned or is currently being patterned into distinct domains of gene expression. How such tissue patterns are maintained during the large scale tissue movements of convergent extension has thus far not been investigated. Intriguingly, experimental data indicate that in certain cases these tissue patterns may drive convergent extension rather than requiring safeguarding against convergent extension. Here we use a 2D Cellular Potts Model (CPM) of a tissue prepatterned into segments, to show that convergent extension tends to disrupt this pre-existing segmental pattern. However, when cells preferentially adhere to cells of the same segment type, segment integrity is maintained without any reduction in tissue extension. Strikingly, we demonstrate that this segment-specific adhesion is by itself sufficient to drive convergent extension. Convergent extension is enhanced when we endow our in silico cells with persistence of motion, which in vivo would naturally follow from cytoskeletal dynamics. Finally, we extend our model to confirm the generality of our results. We demonstrate a similar effect of differential adhesion on convergent extension in tissues that can only extend in a single direction (as often occurs due to the inertia of the head region of the embryo), and in tissues prepatterned into a sequence of domains resulting in two opposing adhesive gradients, rather than alternating segments.

A coordinated switch in sucrose and callose metabolism enables enhanced symplastic unloading in potato tubers.

One of the early changes upon tuber induction is the switch from apoplastic to symplastic unloading. Whether and how this change in unloading mode contributes to sink strength has remained unclear. In addition, developing tubers also change from energy to storage-based sucrose metabolism. Here, we investigated the coordination between changes in unloading mode and sucrose metabolism and their relative role in tuber sink strength by looking into callose and sucrose metabolism gene expression combined with a model of apoplastic and symplastic unloading. Gene expression analysis suggests that callose deposition in tubers is decreased by lower callose synthase expression. Furthermore, changes in callose and sucrose metabolism are strongly correlated, indicating a well-coordinated developmental switch. Modelling indicates that symplastic unloading is not the most efficient unloading mode per se. Instead, it is the concurrent metabolic switch that provides the physiological conditions necessary to potentiate symplastic transport and thereby enhance tuber sink strength .

Transport properties of canonical PIN-FORMED proteins from Arabidopsis and the role of the loop domain in auxin transport.

The phytohormone auxin is polarly transported in plants by PIN-FORMED (PIN) transporters and controls virtually all growth and developmental processes. Canonical PINs possess a long, largely disordered cytosolic loop. Auxin transport by canonical PINs is activated by loop phosphorylation by certain kinases. The structure of the PIN transmembrane domains was recently determined, their transport properties remained poorly characterized, and the role of the loop in the transport process was unclear. Here, we determined the quantitative kinetic parameters of auxin transport mediated by Arabidopsis PINs to mathematically model auxin distribution in roots and to test these predictions in vivo. Using chimeras between transmembrane and loop domains of different PINs, we demonstrate a strong correlation between transport parameters and physiological output, indicating that the loop domain is not only required to activate PIN-mediated auxin transport, but it has an additional role in the transport process by a currently unknown mechanism.

Parallels between drought and flooding: An integrated framework for plant eco-physiological responses to water stress.

Drought and flooding occur at opposite ends of the soil moisture spectrum yet their resulting stress responses in plants share many similarities. Drought limits root water uptake to which plants respond with stomatal closure and reduced leaf gas exchange. Flooding limits root metabolism due to soil oxygen deficiency, which also limits root water uptake and leaf gas exchange. As drought and flooding can occur consecutively in the same system and resulting plant stress responses share similar mechanisms, a single theoretical framework that integrates plant responses over a continuum of soil water conditions from drought to flooding is attractive. Based on a review of recent literature, we integrated the main plant eco-physiological mechanisms in a single theoretical framework with a focus on plant water transport, plant oxygen dynamics, and leaf gas exchange. We used theory from the soil-plant-atmosphere continuum modeling as "backbone" for our framework, and subsequently incorporated interactions between processes that regulate plant water and oxygen status, abscisic acid and ethylene levels, and the resulting acclimation strategies in response to drought, waterlogging, and complete submergence. Our theoretical framework provides a basis for the development of mathematical models to describe plant responses to the soil moisture continuum from drought to flooding.

Evolution of networks for body plan patterning; interplay of modularity, robustness and evolvability.

A major goal of evolutionary developmental biology (evo-devo) is to understand how multicellular body plans of increasing complexity have evolved, and how the corresponding developmental programs are genetically encoded. It has been repeatedly argued that key to the evolution of increased body plan complexity is the modularity of the underlying developmental gene regulatory networks (GRNs). This modularity is considered essential for network robustness and evolvability. In our opinion, these ideas, appealing as they may sound, have not been sufficiently tested. Here we use computer simulations to study the evolution of GRNs' underlying body plan patterning. We select for body plan segmentation and differentiation, as these are considered to be major innovations in metazoan evolution. To allow modular networks to evolve, we independently select for segmentation and differentiation. We study both the occurrence and relation of robustness, evolvability and modularity of evolved networks. Interestingly, we observed two distinct evolutionary strategies to evolve a segmented, differentiated body plan. In the first strategy, first segments and then differentiation domains evolve (SF strategy). In the second scenario segments and domains evolve simultaneously (SS strategy). We demonstrate that under indirect selection for robustness the SF strategy becomes dominant. In addition, as a byproduct of this larger robustness, the SF strategy is also more evolvable. Finally, using a combined functional and architectural approach, we determine network modularity. We find that while SS networks generate segments and domains in an integrated manner, SF networks use largely independent modules to produce segments and domains. Surprisingly, we find that widely used, purely architectural methods for determining network modularity completely fail to establish this higher modularity of SF networks. Finally, we observe that, as a free side effect of evolving segmentation and differentiation in combination, we obtained in-silico developmental mechanisms resembling mechanisms used in vertebrate development.

Embodiment in distributed information processing: "Solid" plants versus "liquid" ant colonies.

Information processing is an essential part of biology, enabling coordination of intra-organismal processes such as development, environmental adaptation and inter-organismal communication. Whilst in animals with specialised brain tissue a substantial amount of information processing occurs in a centralised manner, most biological computing is distributed across multiple entities, such as cells in a tissue, roots in a root system or ants in a colony. Physical context, called embodiment, also affects the nature of biological computing. While plants and ant colonies both perform distributed computing, in plants the units occupy fixed positions while individual ants move around. This distinction, solid versus liquid brain computing, shapes the nature of computations. Here we compare information processing in plants and ant colonies, highlighting how similarities and differences originate in, as well as make use of, the differences in embodiment. We end with a discussion on how this embodiment perspective may inform the debate on plant cognition.