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Arabidopsis possesses several genes related to the multidrug resistance (MDR) genes of animals, one of which, AtMDR1, was shown to be induced by the hormone auxin. Plants having mutations in AtMDR1 or its closest relative, AtPGP1, were isolated by a reverse genetic strategy. Auxin transport activity was greatly impaired in atmdr1 and atmdr1 atpgp1 double mutant plants. Epinastic cotyledons and reduced apical dominance were mutant phenotypes consistent with the disrupted basipetal flow of auxin. The auxin transport inhibitor 1-naphthylphthalamic acid was shown to bind tightly and specifically to AtMDR1 and AtPGP1 proteins. The results indicate that these two MDR-like genes of Arabidopsis encode 1-naphthylphthalamic acid binding proteins that are required for normal auxin distribution and auxin-mediated development.

1. Auxin (IAA) transport was investigated using crown gall suspension tissue culture cells. We have shown that auxin can cross the plasmalemma both by transport of IAA anions on a saturable carrier and by passive (not carriermediated) diffusion of the lipid-soluble undissociated IAA molecules (pK=4.7). The pH optimum of the carrier for auxin influx is about pH 6 and it is half-saturated by auxin concentrations in the region of 1–5 μM. We found that the synthetic auxin 2,4D specifically inhibited carrier-mediated IAA anion influx, and possibly also efflux. Other lipid-soluble weak acids which are not auxins, such as 3,4-dichlorobenzoic acid, had no effect on auxin transport. By contrast, we found that TIBA, an inhibitor of polar auxin transport in intact tissues inhibited only the carrier-mediated efflux of IAA. 2. When the pH outside the cells is maintained below that of the cytoplasm (pH 7), auxin can be accumulated by the cells: In the initial phase of uptake, the direction of the auxin concentration gradient allows both passive carrier-mediated anion influx (inhibited by 2,4D) and a passive diffusion of undissociated acid molecules into the cells. Once inside the cytoplasm, the undissociated molecules ionise, producing IAA anions, to a greater extent than in the more acidic extracellular environment. Uptake by passive diffusion continues as long as the extracellular concentration of undissociated acid remains higher than its intra-cellular concentration. Thus, the direction of the auxin anion concentration gradient is reversed after a short period of uptake and auxin accumulates within the cells. The carrier is now able to mediate passive IAA anion efflux (inhibited by TIBA) down this concentration gradient even though net uptake still proceeds because the carrier is saturable whereas passive diffusion is not. 3. Auxin “secretion” from cells is regarded as a critical step in polar auxin transport. The evidence which we present is consistent with the view that auxin “secretion” depends on a passive carrier-mediated efflux of auxin anions which accumulate within the cells when the extra-cellular pH is below that of the cytoplasm. The implications of this view for theories of polar auxin transport are discussed.

SUMMARY The distribution of IAA between the vacuole and the bathing solution in Hydrodictyon africanum is consistent with passive entry of undissociated IAA and passive efflux of both undissociated IAA and of IAA-, with PIAA (permeability coefficient)* about io-3 cms-1 and PIAA- about -6 -1A o-6 cm s . The involvement of IAA- in the efflux results from the inside-negative P.D. between the medium and the vacuole. The cytoplasm is at a higher pH than either the vacuole or the bathing solution used in most experiments; this is maintained by active H+ efflux at the plasmalemma, and active influx at the tonoplast. In this situation the efflux of IAA- is further promoted by the increased concentration of IAA- in the relatively alkaline cytoplasm. Thus the apparent active efflux of IAA from these cells can be explained in terms of passive driving forces of concentration and electrical potential acting on IAA and IAA-, with the distribution of these two species dictated ultimately by PIAA, PIAA-) the pH of the various compartments and the electrical potential difference between them. If PIAA/PIAA- were larger at the apical than at the basal end of coleoptile cells, such an effect could explain polar IAA transport, with metabolic energy being used only to maintain the relative permeabilities to the two species, the pH gradient and the electrical gradient.

SummarySpatial distribution of the plant hormone auxin regulates multiple aspects of plant development. These self-regulating auxin gradients are established by the action of PIN auxin transporters, whose activity is regulated by their constitutive cycling between the plasma membrane and endosomes. Here, we show that auxin signaling by the auxin receptor AUXIN-BINDING PROTEIN 1 (ABP1) inhibits the clathrin-mediated internalization of PIN proteins. ABP1 acts as a positive factor in clathrin recruitment to the plasma membrane, thereby promoting endocytosis. Auxin binding to ABP1 interferes with this action and leads to the inhibition of clathrin-mediated endocytosis. Our study demonstrates that ABP1 mediates a nontranscriptional auxin signaling that regulates the evolutionarily conserved process of clathrin-mediated endocytosis and suggests that this signaling may be essential for the developmentally important feedback of auxin on its own transport.

The pattern of plant organ initiation at the shoot apical meristem (SAM), termed phyllotaxis, displays regularities that have long intrigued botanists and mathematicians alike. In the SAM, the central zone (CZ) contains a population of stem cells that replenish the surrounding peripheral zone (PZ), where organs are generated in regular patterns. These patterns differ between species and may change in response to developmental or environmental cues [1]. Expression analysis of auxin efflux facilitators of the PIN-FORMED (PIN) family combined with modeling of auxin transport has indicated that organ initiation is associated with intracellular polarization of PIN proteins and auxin accumulation [2-10]. However, regulators that modulate PIN activity to determine phyllotactic patterns have hitherto been unknown. Here we reveal that three redundantly acting PLETHORA (PLT)-like AP2 domain transcription factors control shoot organ positioning in the model plant Arabidopsis thaliana. Loss of PLT3, PLT5, and PLT7 function leads to nonrandom, metastable changes in phyllotaxis. Phyllotactic changes in plt3plt5plt7 mutants are largely attributable to misregulation of PIN1 and can be recapitulated by reducing PIN1 dosage, revealing that PLT proteins are key regulators of PIN1 activity in control of phyllotaxis. © 2011 Elsevier Ltd. All rights reserved.

Arabidopsis plants carrying mutations in the PINOID (PID) gene have a pleiotropic shoot phenotype that mimics that of plants grown on auxin transport inhibitors or of plants mutant for the auxin efflux carrier PINFORMED (PIN), with defects in the formation of cotyledons, flowers, and floral organs. We have cloned PID and find that it is transiently expressed in the embryo and in initiating floral anlagen, demonstrating a specific role for PID in promoting primordium development. Constitutive expression of PID causes a phenotype in both shoots and roots that is similar to that of auxin-insensitive plants, implying that PID, which encodes a serine-threonine protein kinase, negatively regulates auxin signaling.

Timing of leaf abscission is an important trait for biomass production and seasonal acclimation in deciduous trees. The signaling leading to organ separation, from the external cue (decreasing photoperiod) to ethylene-regulated hydrolysis of the middle lamellae in the abscission zone, is only poorly understood. Data from annual species indicate that the formation of an auxin gradient spanning the abscission zone regulates the timing of abscission. We established an experimental system in Populus to induce leaf shedding synchronously under controlled greenhouse conditions in order to test the function of auxin in leaf abscission. Here, we show that exogenous auxin delayed abscission of dark-induced leaves over short and long distances and that a new auxin response maximum preceded the formation of an abscission zone. Several auxin transporters were down-regulated during abscission and inhibition of polar auxin transport delayed leaf shedding. Ethylene signaling was not involved in the regulation of these auxin transporters and in the formation of an abscission zone, but was required for the expression of hydrolytic enzymes associated with cell separation. Since exogenous auxin delayed abscission in absence of ethylene signaling auxin likely acts independently of ethylene signaling on cell separation.

Auxin directs plant ontogenesis via differential accumulation within tissues depending largely on the activity of PIN proteins that mediate auxin efflux from cells and its directional cell-to-cell transport. Regardless of the developmental importance of PINs, the structure of these transporters is poorly characterized. Here, we present experimental data concerning protein topology of plasma membrane-localized PINs. Utilizing approaches based on pH-dependent quenching of fluorescent reporters combined with immunolocalization techniques, we mapped the membrane topology of PINs and further cross-validated our results using available topology modeling software. We delineated the topology of PIN1 with two transmembrane (TM) bundles of five α-helices linked by a large intracellular loop and a C-terminus positioned outside the cytoplasm. Using constraints derived from our experimental data, we also provide an updated position of helical regions generating a verisimilitude model of PIN1. Since the canonical long PINs show a high degree of conservation in TM domains and auxin transport capacity has been demonstrated for Arabidopsis representatives of this group, this empirically enhanced topological model of PIN1 will be an important starting point for further studies on PIN structure-function relationships. In addition, we have established protocols that can be used to probe the topology of other plasma membrane proteins in plants.

International audience; Lateral roots originate deep within the parental root from a small number of founder cells at the periphery of vascular tissues and must emerge through intervening layers of tissues. We describe how the hormone auxin, which originates from the developing lateral root, acts as a local inductive signal which re-programmes adjacent cells. Auxin induces the expression of a previously uncharacterized auxin influx carrier LAX3 in cortical and epidermal cells directly overlaying new primordia. Increased LAX3 activity reinforces the auxin-dependent induction of a selection of cell-wall-remodelling enzymes, which are likely to promote cell separation in advance of developing lateral root primordia.

• Patterning in Arabidopsis root development is coordinated via a localized auxin concentration maximum in the root tip, requiring the regulated expression of specific genes. However, little is known about how hormone and gene expression patterning is generated. • Using a variety of experimental data, we develop a spatiotemporal hormonal crosstalk model that describes the integrated action of auxin, ethylene and cytokinin signalling, the POLARIS protein, and the functions of PIN and AUX1 auxin transporters. We also conduct novel experiments to confirm our modelling predictions. • The model reproduces auxin patterning and trends in wild-type and mutants; reveals that coordinated PIN and AUX1 activities are required to generate correct auxin patterning; correctly predicts shoot to root auxin flux, auxin patterning in the aux1 mutant, the amounts of cytokinin, ethylene and PIN protein, and PIN protein patterning in wild-type and mutant roots. Modelling analysis further reveals how PIN protein patterning is related to the POLARIS protein through ethylene signalling. Modelling prediction of the patterning of POLARIS expression is confirmed experimentally. • Our combined modelling and experimental analysis reveals that a hormonal crosstalk network regulates the emergence of patterns and levels of hormones and gene expression in wild-type and mutants.

The plant hormone auxin is a conserved regulator of development which has been implicated in the generation of morphological novelty. PIN-FORMED1 (PIN) auxin efflux carriers are central to auxin function by regulating its distribution. PIN family members have divergent structures and cellular localizations, but the origin and evolutionary significance of this variation is unresolved. To characterize PIN family evolution, we have undertaken phylogenetic and structural analyses with a massive increase in taxon sampling over previous studies. Our phylogeny shows that following the divergence of the bryophyte and lycophyte lineages, two deep duplication events gave rise to three distinct lineages of PIN proteins in euphyllophytes. Subsequent independent radiations within each of these lineages were taxonomically asymmetric, giving rise to at least 21 clades of PIN proteins, of which 15 are revealed here for the first time. Although most PIN protein clades share a conserved canonical structure with a modular central loop domain, a small number of noncanonical clades dispersed across the phylogeny have highly divergent protein structure. We propose that PIN proteins underwent sub- and neofunctionalization with substantial modification to protein structure throughout plant evolution. Our results have important implications for plant evolution as they suggest that structurally divergent PIN proteins that arose in paralogous radiations contributed to the convergent evolution of organ systems in different land plant lineages.

Summary Background Plant body plans arise by the activity of meristematic growing tips during development and radiated independently in the gametophyte (n) and sporophyte (2n) stages of the life cycle during evolution. Although auxin and its intercellular transport by PIN family efflux carriers are primary regulators of sporophytic shoot development in flowering plants, the extent of conservation in PIN function within the land plants and the mechanisms regulating bryophyte gametophytic shoot development are largely unknown. Results We have found that treating gametophytic shoots of the moss Physcomitrella patens with exogenous auxins and auxin transport inhibitors disrupts apical function and leaf development. Two plasma membrane-targeted PIN proteins are expressed in leafy shoots, and pin mutants resemble plants treated with auxins or auxin transport inhibitors. PIN-mediated auxin transport regulates apical cell function, leaf initiation, leaf shape, and shoot tropisms in moss gametophytes. pin mutant sporophytes are sometimes branched, reproducing a phenotype only previously seen in the fossil record and in rare natural moss variants. Conclusions Our results show that PIN-mediated auxin transport is an ancient, conserved regulator of shoot development.

The epidermis is hypothesized to play a signalling role during plant development. One class of mutants showing defects in signal transduction and radial patterning are those in sterol biosynthesis. The expectation is that living cells require sterols, but it is not clear that all cell types express sterol biosynthesis genes. The HYDRA1 (HYD1) gene of Arabidopsis encodes sterol Δ8-Δ7 isomerase, and although hyd1 seedlings are defective in radial patterning across several tissues, we show that the HYD1 gene is expressed most strongly in the root epidermis. Transgenic activation of HYD1 transcription in the epidermis of hyd1 null mutants reveals a major role in root patterning and growth. HYD1 expression in the vascular tissues and root meristem, though not endodermis or pericycle, also leads to some phenotypic rescue. Phenotypic rescue is associated with rescued patterning of the PIN1 and PIN2 auxin efflux carriers. The importance of the epidermis in controlling root growth and development is proposed to be in part due to its role as a site for sterol biosynthesis, and auxin is a candidate for the non-cell autonomous signal.

Understanding the mechanisms regulating root development under drought conditions is an important question for plant biology and world agriculture. We examine the effect of osmotic stress on abscisic acid (ABA), cytokinin and ethylene responses and how they mediate auxin transport, distribution and root growth through effects on PIN proteins. We integrate experimental data to construct hormonal crosstalk networks to formulate a systems view of root growth regulation by multiple hormones. Experimental analysis shows: that ABA-dependent and ABA-independent stress responses increase under osmotic stress, but cytokinin responses are only slightly reduced; inhibition of root growth under osmotic stress does not require ethylene signalling, but auxin can rescue root growth and meristem size; osmotic stress modulates auxin transporter levels and localization, reducing root auxin concentrations; PIN1 levels are reduced under stress in an ABA-dependent manner, overriding ethylene effects; and the interplay among ABA, ethylene, cytokinin and auxin is tissue-specific, as evidenced by differential responses of PIN1 and PIN2 to osmotic stress. Combining experimental analysis with network construction reveals that ABA regulates root growth under osmotic stress conditions via an interacting hormonal network with cytokinin, ethylene and auxin.

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.

SummaryIn plants, auxin is a key regulator of development and is unique among plant hormones in that its function requires polarized transport between neighboring cells to form concentration gradients across various plant tissues [1–5]. Although putative auxin-influx [6, 7] and -efflux [8–15] transporters have been identified by using molecular genetic approaches, a detailed functional understanding for many of these transporters remains undetermined. Here we present the functional characterization of the auxin-influx carrier AUX1. Upon expression of AUX1 in Xenopus oocytes, saturable, pH-dependent uptake of 3H-IAA was measured. Mutations in AUX1 that abrogate physiological responses to IAA in planta resulted in loss or reduction of 3H-IAA uptake in AUX1-expressing oocytes. AUX1-mediated uptake of 3H-IAA was reduced by the IAA analogs 2,4-D and 1-NOA, but not by other auxin analogs. The measured Km for AUX1-mediated uptake of 3H-IAA was at concentrations at which physiological responses are observed for exogenously added IAA and 2,4-D. This is the first report demonstrating detailed functional characteristics of a plant auxin-influx transporter. This biochemical characterization provides new insights and a novel tool for studying auxin entry into cells and its pivotal roles in plant growth and development.