Mathematical Modelling of Auxin Transport in Plant Tissues:Flux Meets Signalling and Growth

Plant hormone auxin has critical roles in plant growth, dependent on its heterogeneous distribution in plant tissues. Exactly how auxin transport and developmental processes such as growth coordinate to achieve the precise patterns of auxin observed experimentally is not well understood. Here we use mathematical modelling to examine the interplay between auxin dynamics and growth and their contribution to formation of patterns in auxin distribution in plant tissues. Mathematical models describing the auxin-related signalling pathway, PIN and AUX1 dynamics, auxin transport, and cell growth in plant tissues are derived. A key assumption of our models is the regulation of PIN proteins by the auxin-responsive ARF-Aux/IAA signalling pathway, with upregulation of PIN biosynthesis by ARFs. Models are analysed and solved numerically to examine the long-time behaviour and auxin distribution. Changes in auxin-related signalling processes are shown to be able to trigger transition between passage and spot type patterns in auxin distribution. The model was also shown to be able to generate isolated cells with oscillatory dynamics in levels of components of the auxin signalling pathway which could explain oscillations in levels of ARF targets that have been observed experimentally. Cell growth was shown to have influence on PIN polarisation and determination of auxin distribution patterns. Numerical simulation results indicate that auxin-related signalling processes can explain the different patterns in auxin distributions observed in plant tissues, whereas the interplay between auxin transport and growth can explain the `reverse-fountain' pattern in auxin distribution observed at plant root tips

Hormonal interactions in the control of Arabidopsis hypocotyl elongation

The Arabidopsis hypocotyl, together with hormone mutants and chemical inhibitors, was used to study the role of auxin iri cell elongation and its possible interactions with ethylene and gibberellin. When wild-type Arabidopsis seedlings were grown on media containing a range of auxin concentrations, hypocotyl growth was inhibited. However, when axr1-12 and 35S-iaaL (which have reduced auxin response and levels, respectively) were grown in the same conditions, auxin was able to promote hypocotyl growth. In contrast, auxin does not promote hypocotyl growth of axr3-1, which has phenotypes that suggest an enhanced auxin response. These results are consistent with the hypothesis that auxin levels in the wild-type hypocotyl are optimal for elongation and that additional auxin is inhibitory. When ethylene responses were reduced using either the ethylene-resistant mutant etr1 or aminoethoxyvinylglycine, an inhibitor of ethylene synthesis, auxin responses were unchanged, indicating that auxin does not inhibit hypocotyl elongation through ethylene. To test for interactions between auxin and gibberellin, auxin mutants were grown on media containing gibberellin and gibberellin mutants were grown on media containing auxin. The responses were found to be the same as wild-type Arabidopsis seedlings in all cases. In addition, 1 muM of the auxin transport inhibitor 1-naphthylphthalmic acid does not alter the response of wild-type seedlings to gibberellin. Double mutants were made between gibberellin and auxin mutants and the phenotypes of these appear additive. These results indicate that auxin and gibberellin are acting independently in hypocotyl elongation. Thus auxin, ethylene, and gibberellin each regulate hypocotyl elongation independently

Single-cell-based system to monitor carrier driven cellular auxin homeostasis

Background: Abundance and distribution of the plant hormone auxin play important roles in plant development. Besides other metabolic processes, various auxin carriers control the cellular level of active auxin and, hence, are major regulators of cellular auxin homeostasis. Despite the developmental importance of auxin transporters, a simple medium-to-high throughput approach to assess carrier activities is still missing. Here we show that carrier driven depletion of cellular auxin correlates with reduced nuclear auxin signaling in tobacco Bright Yellow-2 (BY-2) cell cultures. Results: We developed an easy to use transient single-cell-based system to detect carrier activity. We use the relative changes in signaling output of the auxin responsive promoter element DR5 to indirectly visualize auxin carrier activity. The feasibility of the transient approach was demonstrated by pharmacological and genetic interference with auxin signaling and transport. As a proof of concept, we provide visual evidence that the prominent auxin transport proteins PIN-FORMED (PIN) 2 and PIN5 regulate cellular auxin homeostasis at the plasma membrane and endoplasmic reticulum (ER), respectively. Our data suggest that PIN2 and PIN5 have different sensitivities to the auxin transport inhibitor 1-naphthylphthalamic acid (NPA). Also the putative PIN-LIKES (PILS) auxin carrier activity at the ER is insensitive to NPA in our system, indicating that NPA blocks intercellular, but not intracellular auxin transport. Conclusions: This single-cell-based system is a useful tool by which the activity of putative auxin carriers, such as PINs, PILS and WALLS ARE THIN1 (WAT1), can be indirectly visualized in a medium-to-high throughput manner. Moreover, our single cell system might be useful to investigate also other hormonal signaling pathways, such as cytokinin

Combined in silico/in vivo analysis of mechanisms providing for root apical meristem self-organization and maintenance.

Background and aimsThe root apical meristem (RAM) is the plant stem cell niche which provides for the formation and continuous development of the root. Auxin is the main regulator of RAM functioning, and auxin maxima coincide with the sites of RAM initiation and maintenance. Auxin gradients are formed due to local auxin biosynthesis and polar auxin transport. The PIN family of auxin transporters plays a critical role in polar auxin transport, and two mechanisms of auxin maximum formation in the RAM based on PIN-mediated auxin transport have been proposed to date: the reverse fountain and the reflected flow mechanisms.MethodsThe two mechanisms are combined here in in silico studies of auxin distribution in intact roots and roots cut into two pieces in the proximal meristem region. In parallel, corresponding experiments were performed in vivo using DR5::GFP Arabidopsis plants.Key resultsThe reverse fountain and the reflected flow mechanism naturally cooperate for RAM patterning and maintenance in intact root. Regeneration of the RAM in decapitated roots is provided by the reflected flow mechanism. In the excised root tips local auxin biosynthesis either alone or in cooperation with the reverse fountain enables RAM maintenance.ConclusionsThe efficiency of a dual-mechanism model in guiding biological experiments on RAM regeneration and maintenance is demonstrated. The model also allows estimation of the concentrations of auxin and PINs in root cells during development and under various treatments. The dual-mechanism model proposed here can be a powerful tool for the study of several different aspects of auxin function in root

A combinatorial TIR1/AFB–Aux/IAA co-receptor system for differential sensing of auxin

The plant hormone auxin regulates virtually every aspect of plant growth and development. Auxin acts by binding the F-box protein transport inhibitor response 1 (TIR1) and promotes the degradation of the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) transcriptional repressors. Here we show that efficient auxin binding requires assembly of an auxin co-receptor complex consisting of TIR1 and an Aux/IAA protein. Heterologous experiments in yeast and quantitative IAA binding assays using purified proteins showed that different combinations of TIR1 and Aux/IAA proteins form co-receptor complexes with a wide range of auxin-binding affinities. Auxin affinity seems to be largely determined by the Aux/IAA. As there are 6 TIR1/AUXIN SIGNALING F-BOX proteins (AFBs) and 29 Aux/IAA proteins in Arabidopsis thaliana, combinatorial interactions may result in many co-receptors with distinct auxin-sensing properties. We also demonstrate that the AFB5–Aux/IAA co-receptor selectively binds the auxinic herbicide picloram. This co-receptor system broadens the effective concentration range of the hormone and may contribute to the complexity of auxin response

Redirection of auxin flow in Arabidopsis thaliana roots after infection by root-knot nematodes

Plant auxin efflux and influx proteins redirect the plant hormone auxin towards the feeding site upon root-knot nematode infection in Arabidopsis thaliana roots.Plant-parasitic root-knot nematodes induce the formation of giant cells within the plant root, and it has been recognized that auxin accumulates in these feeding sites. Here, we studied the role of the auxin transport system governed by AUX1/LAX3 influx proteins and different PIN efflux proteins during feeding site development in Arabidopsis thaliana roots. Data generated via promoter-reporter line and protein localization analyses evoke a model in which auxin is being imported at the basipetal side of the feeding site by the concerted action of the influx proteins AUX1 and LAX3, and the efflux protein PIN3. Mutants in auxin influx proteins AUX1 and LAX3 bear significantly fewer and smaller galls, revealing that auxin import into the feeding sites is needed for their development and expansion. The feeding site development in auxin export (PIN) mutants was only slightly hampered. Expression of some PINs appears to be suppressed in galls, probably to prevent auxin drainage. Nevertheless, a functional PIN4 gene seems to be a prerequisite for proper nematode development and gall expansion, most likely by removing excessive auxin to stabilize the hormone level in the feeding site. Our data also indicate a role of local auxin peaks in nematode attraction towards the root

Auxin regulates SCFTIR1-dependent degradation of AUX/IAA proteins

The plant hormone auxin is central in many aspects of plant development. Previous studies have implicated the ubiquitin-ligase SCFTIR1 and the AUX/IAA proteins in auxin response. Dominant mutations in several AUX/IAA genes confer pleiotropic auxin-related phenotypes, whereas recessive mutations affecting the function of SCFTIR1 decrease auxin response. Here we show that SCFTIR1 is required for AUX/IAA degradation. We demonstrate that SCFTIR1 interacts with AXR2/IAA7 and AXR3/IAA17, and that domain II of these proteins is necessary and sufficient for this interaction. Further, auxin stimulates binding of SCFTIR1 to the AUX/IAA proteins, and their degradation. Because domain II is conserved in nearly all AUX/IAA proteins in Arabidopsis, we propose that auxin promotes the degradation of this large family of transcriptional regulators, leading to diverse downstream effects

Molecular genetics of auxin signaling

The plant hormone auxin is a simple molecule similar to tryptophan, yet it elicits a diverse array of responses and is involved in the regulation of growth and development throughout the plant life cycle. The ability of auxin to bring about such diverse responses appears to result partly from the existence of several independent mechanisms for auxin perception. Furthermore, one prominent mechanism for auxin signal transduction involves the targeted degradation of members of a large family of transcriptional regulators that appear to participate in complex and competing dimerization networks to modulate the expression of a wide range of genes. These models for auxin signaling now offer a framework in which to test how each specific response to auxin is brought about

Subcellular trafficking of the Arabidopsis auxin influx carrier AUX1 uses a novel pathway distinct from PIN1

The directional flow of the plant hormone auxin mediates multiple developmental processes, including patterning and tropisms. Apical and basal plasma membrane localization of AUXIN-RESISTANT1 (AUX1) and PIN-FORMED1 (PIN1) auxin transport components underpins the directionality of intercellular auxin flow in Arabidopsis thaliana roots. Here, we examined the mechanism of polar trafficking of AUX1. Real-time live cell analysis along with subcellular markers revealed that AUX1 resides at the apical plasma membrane of protophloem cells and at highly dynamic subpopulations of Golgi apparatus and endosomes in all cell types. Plasma membrane and intracellular pools of AUX1 are interconnected by actin-dependent constitutive trafficking, which is not sensitive to the vesicle trafficking inhibitor brefeldin A. AUX1 subcellular dynamics are not influenced by the auxin influx inhibitor NOA but are blocked by the auxin efflux inhibitors TIBA and PBA. Furthermore, auxin transport inhibitors and interference with the sterol composition of membranes disrupt polar AUX1 distribution at the plasma membrane. Compared with PIN1 trafficking, AUX1 dynamics display different sensitivities to trafficking inhibitors and are independent of the endosomal trafficking regulator ARF GEF GNOM. Hence, AUX1 uses a novel trafficking pathway in plants that is distinct from PIN trafficking, providing an additional mechanism for the fine regulation of 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