Krzysztof Wabnik

Recycling, clustering, and endocytosis jointly maintain PIN auxin carrier polarity at the plasma membrane.

Cell polarity reflected by asymmetric distribution of proteins at the plasma membrane is a fundamental feature of unicellular and multicellular organisms. It remains conceptually unclear how cell polarity is kept in cell wall‐encapsulated plant cells. We have used super‐resolution and semi‐quantitative live‐cell imaging in combination with pharmacological, genetic, and computational approaches to reveal insights into the mechanism of cell polarity maintenance in Arabidopsis thaliana. We show that polar‐competent PIN transporters for the phytohormone auxin are delivered to the center of polar domains by super‐polar recycling. Within the plasma membrane, PINs are recruited into non‐mobile membrane clusters and their lateral diffusion is dramatically reduced, which ensures longer polar retention. At the circumventing edges of the polar domain, spatially defined internalization of escaped cargos occurs by clathrin‐dependent endocytosis. Computer simulations confirm that the combination of these processes provides a robust mechanism for polarity maintenance in plant cells. Moreover, our study suggests that the regulation of lateral diffusion and spatially defined endocytosis, but not super‐polar exocytosis have primary importance for PIN polarity maintenance.

Emergence of tissue polarization from synergy of intracellular and extracellular auxin signaling

Plant development is exceptionally flexible as manifested by its potential for organogenesis and regeneration, which are processes involving rearrangements of tissue polarities. Fundamental questions concern how individual cells can polarize in a coordinated manner to integrate into the multicellular context. In canalization models, the signaling molecule auxin acts as a polarizing cue, and feedback on the intercellular auxin flow is key for synchronized polarity rearrangements. We provide a novel mechanistic framework for canalization, based on up‐to‐date experimental data and minimal, biologically plausible assumptions. Our model combines the intracellular auxin signaling for expression of PINFORMED (PIN) auxin transporters and the theoretical postulation of extracellular auxin signaling for modulation of PIN subcellular dynamics. Computer simulations faithfully and robustly recapitulated the experimentally observed patterns of tissue polarity and asymmetric auxin distribution during formation and regeneration of vascular systems and during the competitive regulation of shoot branching by apical dominance. Additionally, our model generated new predictions that could be experimentally validated, highlighting a mechanistically conceivable explanation for the PIN polarization and canalization of the auxin flow in plants.

WOX5-IAA17 Feedback Circuit-Mediated Cellular Auxin Response Is Crucial for the Patterning of Root Stem Cell Niches in Arabidopsis.

In plants, the patterning of stem cell-enriched meristems requires a graded auxin response maximum that emerges from the concerted action of polar auxin transport, auxin biosynthesis, auxin metabolism, and cellular auxin response machinery. However, mechanisms underlying this auxin response maximum-mediated root stem cell maintenance are not fully understood. Here, we present unexpected evidence that WUSCHEL-RELATED HOMEOBOX 5 (WOX5) transcription factor modulates expression of auxin biosynthetic genes in the quiescent center (QC) of the root and thus provides a robust mechanism for the maintenance of auxin response maximum in the root tip. This WOX5 action is balanced through the activity of indole-3-acetic acid 17 (IAA17) auxin response repressor. Our combined genetic, cell biology, and computational modeling studies revealed a previously uncharacterized feedback loop linking WOX5-mediated auxin production to IAA17-dependent repression of auxin responses. This WOX5-IAA17 feedback circuit further assures the maintenance of auxin response maximum in the root tip and thereby contributes to the maintenance of distal stem cell (DSC) populations. Our experimental studies and in silico computer simulations both demonstrate that the WOX5-IAA17 feedback circuit is essential for the maintenance of auxin gradient in the root tip and the auxin-mediated root DSC differentiation.

Modeling Framework for the Establishment of the Apical-Basal Embryonic Axis in Plants

The apical-basal axis of the early plant embryo determines the body plan of the adult organism. To establish a polarized embryonic axis, plants evolved a unique mechanism that involves directional, cell-to-cell transport of the growth regulator auxin. Auxin transport relies on PIN auxin transporters, whose polar subcellular localization determines the flow directionality. PIN-mediated auxin transport mediates the spatial and temporal activity of the auxin response machinery that contributes to embryo patterning processes, including establishment of the apical (shoot) and basal (root) embryo poles. However, little is known of upstream mechanisms guiding the (re)polarization of auxin fluxes during embryogenesis. Here, we developed a model of plant embryogenesis that correctly generates emergent cell polarities and auxin-mediated sequential initiation of apical-basal axis of plant embryo. The model relies on two precisely localized auxin sources and a feedback between auxin and the polar, subcellular PIN transporter localization. Simulations reproduced PIN polarity and auxin distribution, as well as previously unknown polarization events during early embryogenesis. The spectrum of validated model predictions suggests that our model corresponds to a minimal mechanistic framework for initiation and orientation of the apical-basal axis to guide both embryonic and postembryonic plant development.

Prototype cell-to-cell auxin transport mechanism by intracellular auxin compartmentalization

Carrier-dependent, intercellular auxin transport is central to the developmental patterning of higher plants (tracheophytes). The evolution of this polar auxin transport might be linked to the translocation of some PIN auxin efflux carriers from their presumably ancestral localization at the endoplasmic reticulum (ER) to the polar domains at the plasma membrane. Here we propose an eventually ancient mechanism of intercellular auxin distribution by ER-localized auxin transporters involving intracellular auxin retention and switch-like release from the ER. The proposed model integrates feedback circuits utilizing the conserved nuclear auxin signaling for the regulation of PIN transcription and a hypothetical ER-based signaling for the regulation of PIN-dependent transport activity at the ER. Computer simulations of the model revealed its plausibility for generating auxin channels and localized auxin maxima highlighting the possibility of this alternative mechanism for polar auxin transport.

Cytokinin response factors regulate PIN-FORMED auxin transporters

Auxin and cytokinin are key endogenous regulators of plant development. Although cytokinin-mediated modulation of auxin distribution is a developmentally crucial hormonal interaction, its molecular basis is largely unknown. Here we show a direct regulatory link between cytokinin signalling and the auxin transport machinery uncovering a mechanistic framework for cytokinin-auxin cross-talk. We show that the CYTOKININ RESPONSE FACTORS (CRFs), transcription factors downstream of cytokinin perception, transcriptionally control genes encoding PIN-FORMED (PIN) auxin transporters at a specific PIN CYTOKININ RESPONSE ELEMENT (PCRE) domain. Removal of this cis-regulatory element effectively uncouples PIN transcription from the CRF-mediated cytokinin regulation and attenuates plant cytokinin sensitivity. We propose that CRFs represent a missing cross-talk component that fine-tunes auxin transport capacity downstream of cytokinin signalling to control plant development.

Feedback models for polarized auxin transport: an emerging trend

The phytohormone auxin is vital to plant growth and development. A unique property of auxin among all other plant hormones is its cell-to-cell polar transport that requires activity of polarly localized PIN-FORMED (PIN) auxin efflux transporters. Despite the substantial molecular insight into the cellular PIN polarization, the mechanistic understanding for developmentally and environmentally regulated PIN polarization is scarce. The long-standing belief that auxin modulates its own transport by means of a positive feedback mechanism has inspired both experimentalists and theoreticians for more than two decades. Recently, theoretical models for auxin-dependent patterning in plants include the feedback between auxin transport and the PIN protein localization. These computer models aid to assess the complexity of plant development by testing and predicting plausible scenarios for various developmental processes that occur in planta. Although the majority of these models rely on purely heuristic principles, the most recent mechanistic models tentatively integrate biologically testable components into known cellular processes that underlie the PIN polarity regulation. The existing and emerging computational approaches to describe PIN polarization are presented and discussed in the light of recent experimental data on the PIN polar targeting.

Cellular mechanisms for cargo delivery and polarity maintenance at different polar domains in plant cells

The asymmetric localization of proteins in the plasma membrane domains of eukaryotic cells is a fundamental manifestation of cell polarity that is central to multicellular organization and developmental patterning. In plants, the mechanisms underlying the polar localization of cargo proteins are still largely unknown and appear to be fundamentally distinct from those operating in mammals. Here, we present a systematic, quantitative comparative analysis of the polar delivery and subcellular localization of proteins that characterize distinct polar plasma membrane domains in plant cells. The combination of microscopic analyses and computational modeling revealed a mechanistic framework common to diverse polar cargos and underlying the establishment and maintenance of apical, basal, and lateral polar domains in plant cells. This mechanism depends on the polar secretion, constitutive endocytic recycling, and restricted lateral diffusion of cargos within the plasma membrane. Moreover, our observations suggest that polar cargo distribution involves the individual protein potential to form clusters within the plasma membrane and interact with the extracellular matrix. Our observations provide insights into the shared cellular mechanisms of polar cargo delivery and polarity maintenance in plant cells.

A Model of Differential Growth-Guided Apical Hook Formation in Plants

Combined in silico and in vivo approaches identify a minimal mechanism producing auxin gradient-guided differential growth during the establishment of the apical hook in dicot seedlings. Differential cell growth enables flexible organ bending in the presence of environmental signals such as light or gravity. A prominent example of the developmental processes based on differential cell growth is the formation of the apical hook that protects the fragile shoot apical meristem when it breaks through the soil during germination. Here, we combined in silico and in vivo approaches to identify a minimal mechanism producing auxin gradient-guided differential growth during the establishment of the apical hook in the model plant Arabidopsis thaliana. Computer simulation models based on experimental data demonstrate that asymmetric expression of the PIN-FORMED auxin efflux carrier at the concave (inner) versus convex (outer) side of the hook suffices to establish an auxin maximum in the epidermis at the concave side of the apical hook. Furthermore, we propose a mechanism that translates this maximum into differential growth, and thus curvature, of the apical hook. Through a combination of experimental and in silico computational approaches, we have identified the individual contributions of differential cell elongation and proliferation to defining the apical hook and reveal the role of auxin-ethylene crosstalk in balancing these two processes.

Systems approaches to study root architecture dynamics

The plant root system is essential for providing anchorage to the soil, supplying minerals and water, and synthesizing metabolites. It is a dynamic organ modulated by external cues such as environmental signals, water and nutrients availability, salinity and others. Lateral roots (LRs) are initiated from the primary root post-embryonically, after which they progress through discrete developmental stages which can be independently controlled, providing a high level of plasticity during root system formation. Within this review, main contributions are presented, from the classical forward genetic screens to the more recent high-throughput approaches, combined with computer model predictions, dissecting how LRs and thereby root system architecture is established and developed.