WAVY GROWTH Arabidopsis E3 ubiquitin ligases affect apical PIN sorting decisions.

Directionality in the intercellular transport of the plant hormone auxin is determined by polar plasma membrane localization of PIN-FORMED (PIN) auxin transport proteins. However, apart from PIN phosphorylation at conserved motifs, no further determinants explicitly controlling polar PIN sorting decisions have been identified. Here we present Arabidopsis WAVY GROWTH 3 (WAV3) and closely related RING-finger E3 ubiquitin ligases, whose loss-of-function mutants show a striking apical-to-basal polarity switch in PIN2 localization in root meristem cells. WAV3 E3 ligases function as essential determinants for PIN polarity, acting independently from PINOID/WAG-dependent PIN phosphorylation. They antagonize ectopic deposition of de novo synthesized PIN proteins already immediately following completion of cell division, presumably via preventing PIN sorting into basal, ARF GEF-mediated trafficking. Our findings reveal an involvement of E3 ligases in the selective targeting of apically localized PINs in higher plants.

Early stages of legume-rhizobia symbiosis are controlled by ABCG-mediated transport of active cytokinins.

Growing evidence has highlighted the essential role of plant hormones, notably, cytokinins (CKs), in nitrogen-fixing symbiosis, both at early and late nodulation stages1,2. Despite numerous studies showing the central role of CK in nodulation, the importance of CK transport in the symbiosis is unknown. Here, we show the role of ABCG56, a full-size ATP-binding cassette (ABC) transporter in the early stages of the nodulation. MtABCG56 is expressed in roots and nodules and its messenger RNA levels increase upon treatment with symbiotic bacteria, isolated Nod factor and CKs, accumulating within the epidermis and root cortex. MtABCG56 exports bioactive CKs in an ATP-dependent manner over the plasma membrane and its disruption results in an impairment of nodulation. Our data indicate that ABCG-mediated cytokinin transport is important for proper establishment of N-fixing nodules.

Non-steroidal Anti-inflammatory Drugs Target TWISTED DWARF1-Regulated Actin Dynamics and Auxin Transport-Mediated Plant Development.

The widely used non-steroidal anti-inflammatory drugs (NSAIDs) are derivatives of the phytohormone salicylic acid (SA). SA is well known to regulate plant immunity and development, whereas there have been few reports focusing on the effects of NSAIDs in plants. Our studies here reveal that NSAIDs exhibit largely overlapping physiological activities to SA in the model plant Arabidopsis. NSAID treatments lead to shorter and agravitropic primary roots and inhibited lateral root organogenesis. Notably, in addition to the SA-like action, which in roots involves binding to the protein phosphatase 2A (PP2A), NSAIDs also exhibit PP2A-independent effects. Cell biological and biochemical analyses reveal that many NSAIDs bind directly to and inhibit the chaperone activity of TWISTED DWARF1, thereby regulating actin cytoskeleton dynamics and subsequent endosomal trafficking. Our findings uncover an unexpected bioactivity of human pharmaceuticals in plants and provide insights into the molecular mechanism underlying the cellular action of this class of anti-inflammatory compounds.

Auxin-transporting ABC transporters are defined by a conserved D/E-P motif regulated by a prolylisomerase.

The plant hormone auxin must be transported throughout plants in a cell-to-cell manner to affect its various physiological functions. ABCB transporters are critical for this polar auxin distribution, but the regulatory mechanisms controlling their function is not fully understood. The auxin transport activity of ABCB1 was suggested to be regulated by a physical interaction with FKBP42/Twisted Dwarf1 (TWD1), a peptidylprolyl cis-trans isomerase (PPIase), but all attempts to demonstrate such a PPIase activity by TWD1 have failed so far. By using a structure-based approach, we identified several surface-exposed proline residues in the nucleotide binding domain and linker of Arabidopsis ABCB1, mutations of which do not alter ABCB1 protein stability or location but do affect its transport activity. P1008 is part of a conserved signature D/E-P motif that seems to be specific for auxin-transporting ABCBs, which we now refer to as ATAs. Mutation of the acidic residue also abolishes auxin transport activity by ABCB1. All higher plant ABCBs for which auxin transport has been conclusively proven carry this conserved motif, underlining its predictive potential. Introduction of this D/E-P motif into malate importer, ABCB14, increases both its malate and its background auxin transport activity, suggesting that this motif has an impact on transport capacity. The D/E-P1008 motif is also important for ABCB1-TWD1 interactions and activation of ABCB1-mediated auxin transport by TWD1. In summary, our data imply a new function for TWD1 acting as a putative activator of ABCB-mediated auxin transport by cis-trans isomerization of peptidyl-prolyl bonds.

HSP90 and co-chaperones: a multitaskers' view on plant hormone biology.

In order to survive under ever-changing conditions plants must be able to adaptively respond to their environment. Plant hormones and the signaling cross-talk among them play a key role in integrating external and internal cues, enabling the plants to acclimate accordingly. HSP90 and several of its co-chaperones are known as pleiotropic factors involved in the signaling pathways of multiple stress responses, including temperature, drought, and pathogen infection. Recently, hormone receptor components for auxin and jasmonic acid, respectively, have been identified as clients of the HSP90 chaperone system, suggesting a direct HSP90-dependent link to hormone signaling. In this review, we give an overview of the multiple roles of HSP90 and its co-chaperones in plant hormone biology and discuss the largely unexplored targets for signal integration that the activity of these apparent multitaskers may suggest.

A transportome-scale amiRNA-based screen identifies redundant roles of Arabidopsis ABCB6 and ABCB20 in auxin transport.

Transport of signaling molecules is of major importance for regulating plant growth, development, and responses to the environment. A prime example is the spatial-distribution of auxin, which is regulated via transporters to govern developmental patterning. A critical limitation in our ability to identify transporters by forward genetic screens is their potential functional redundancy. Here, we overcome part of this functional redundancy via a transportome, multi-targeted forward-genetic screen using artificial-microRNAs (amiRNAs). We generate a library of 3000 plant lines expressing 1777 amiRNAs, designed to target closely homologous genes within subclades of transporter families and identify, genotype and quantitatively phenotype, 80 lines showing reproducible shoot growth phenotypes. Within this population, we discover and characterize a strong redundant role for the unstudied ABCB6 and ABCB20 genes in auxin transport and response. The unique multi-targeted lines generated in this study could serve as a genetic resource that is expected to reveal additional transporters.

Tomato ATP-Binding Cassette Transporter SlABCB4 Is Involved in Auxin Transport in the Developing Fruit.

Plant ATP binding cassette (ABC) transporters are membrane proteins that are important for transporting a wide range of compounds, including secondary metabolites and phytohormones. In Arabidopsis, some members of the ABCB subfamily of ABC transporter, also known as Multi-Drug Resistance proteins (MDRs), have been implicated in auxin transport. However, reports on the roles of the auxin-mediated ABCBs in fleshy fruit development are rare. Here, we present that SlABCB4, a member of the tomato ABCB subfamily, transports auxin in the developing fruit of tomato. Transient expression of SlABCB4-GFP fusion proteins in tobacco cells showed plasma membrane localization. The transport activity of SlABCB4, expressed in Nicotiana benthamiana protoplasts, revealed substrate specificity for indole-3-acetic acid export. Gene expression analysis of SlABCB4 revealed high expression levels at the early stages of fruit development. Therefore, SlABCB4 is considered to facilitate auxin distribution in tomato fruit, which is important for tomato fruit development.

A Critical View on ABC Transporters and Their Interacting Partners in Auxin Transport.

Different subclasses of ATP-binding cassette (ABC) transporters have been implicated in the transport of native variants of the phytohormone auxin. Here, the putative, individual roles of key members belonging to the ABCB, ABCD and ABCG families, respectively, are highlighted and the knowledge of their assumed expression and transport routes is reviewed and compared with their mutant phenotypes. Protein-protein interactions between ABC transporters and regulatory components during auxin transport are summarized and their importance is critically discussed. There is a focus on the functional interaction between members of the ABCB family and the FKBP42, TWISTED DWARF1, acting as a chaperone during plasma membrane trafficking of ABCBs. Further, the mode and relevance of functional ABCB-PIN interactions is diagnostically re-evaluated. A new nomenclature describing precisely the most likely ABCB-PIN interaction scenarios is suggested. Finally, available tools for the detection and prediction of ABC transporter interactomes are summarized and the potential of future ABC transporter interactome maps is highlighted.

SHADE AVOIDANCE 4 Is Required for Proper Auxin Distribution in the Hypocotyl.

The phytohormone auxin is involved in virtually every aspect of plant growth and development. Through polar auxin transport, auxin gradients can be established, which then direct plant differentiation and growth. Shade avoidance responses are well-known processes that require polar auxin transport. In this study, we have identified a mutant, shade avoidance 4 (sav4), defective in shade-induced hypocotyl elongation and basipetal auxin transport. SAV4 encodes an unknown protein with armadillo repeat- and tetratricopeptide repeat-like domains known to provide protein-protein interaction surfaces. C terminally yellow fluorescent protein-tagged SAV4 localizes to both the plasma membrane and the nucleus. Membrane-localized SAV4 displays a polar association with the shootward plasma membrane domain in hypocotyl and root cells, which appears to be necessary for its function in hypocotyl elongation. Cotransfection of SAV4 and ATP-binding cassette B1 (ABCB1) auxin transporter in tobacco (Nicotiana benthamiana) revealed that SAV4 blocks ABCB1-mediated auxin efflux. We thus propose that polarly localized SAV4 acts to inhibit ABCB-mediated auxin efflux toward shoots and facilitates the establishment of proper auxin gradients.

Plant development regulated by cytokinin sinks.

Morphogenetic signals control the patterning of multicellular organisms. Cytokinins are mobile signals that are perceived by subsets of plant cells. We found that the responses to cytokinin signaling during Arabidopsis development are constrained by the transporter PURINE PERMEASE 14 (PUP14). In our experiments, the expression of PUP14 was inversely correlated to the cytokinin signaling readout. Loss of PUP14 function allowed ectopic cytokinin signaling accompanied by aberrant morphogenesis in embryos, roots, and the shoot apical meristem. PUP14 protein localized to the plasma membrane and imported bioactive cytokinins, thus depleting apoplastic cytokinin pools and inhibiting perception by plasma membrane-localized cytokinin sensors to create a sink for active ligands. We propose that the spatiotemporal cytokinin sink patterns established by PUP14 determine the cytokinin signaling landscape that shapes the morphogenesis of land plants.

TWISTED DWARF1 Mediates the Action of Auxin Transport Inhibitors on Actin Cytoskeleton Dynamics.

Plant growth and architecture is regulated by the polar distribution of the hormone auxin. Polarity and flexibility of this process is provided by constant cycling of auxin transporter vesicles along actin filaments, coordinated by a positive auxin-actin feedback loop. Both polar auxin transport and vesicle cycling are inhibited by synthetic auxin transport inhibitors, such as 1-N-naphthylphthalamic acid (NPA), counteracting the effect of auxin; however, underlying targets and mechanisms are unclear. Using NMR, we map the NPA binding surface on the Arabidopsis thaliana ABCB chaperone TWISTED DWARF1 (TWD1). We identify ACTIN7 as a relevant, although likely indirect, TWD1 interactor, and show TWD1-dependent regulation of actin filament organization and dynamics and that TWD1 is required for NPA-mediated actin cytoskeleton remodeling. The TWD1-ACTIN7 axis controls plasma membrane presence of efflux transporters, and as a consequence act7 and twd1 share developmental and physiological phenotypes indicative of defects in auxin transport. These can be phenocopied by NPA treatment or by chemical actin (de)stabilization. We provide evidence that TWD1 determines downstream locations of auxin efflux transporters by adjusting actin filament debundling and dynamizing processes and mediating NPA action on the latter. This function appears to be evolutionary conserved since TWD1 expression in budding yeast alters actin polarization and cell polarity and provides NPA sensitivity.

Analysis of the role of myosins in targeting proteins to plasmodesmata.

Plasmodesmata (PD) are dynamic cell wall microchannels that facilitate the intercellular trafficking of RNA and protein macromolecules playing cell nonautonomous roles in the orchestration of plant development, growth, and plant defense. The trafficking of macromolecules and organelles within cells depends on cytoskeletal components and their associated motor proteins. Plant viruses evolved to hijack this transport system to move their infectious genomes to PD. Current efforts concentrate on dissecting the role of specific myosin motors in transporting plant or viral proteins to the channels. Here we describe a method that addresses the role of specific myosins by expression of myosin tails that cause the repression of myosin activity in a dominant-negative manner. As an example, we explain the use of myosin tails from Nicotiana benthamiana to address the role of N. benthamiana myosins in the targeting of PLASMODESMATA-LOCATED PROTEIN 1 (PDLP1) to PD.

Myosins VIII and XI play distinct roles in reproduction and transport of tobacco mosaic virus.

Viruses are obligatory parasites that depend on host cellular factors for their replication as well as for their local and systemic movement to establish infection. Although myosin motors are thought to contribute to plant virus infection, their exact roles in the specific infection steps have not been addressed. Here we investigated the replication, cell-to-cell and systemic spread of Tobacco mosaic virus (TMV) using dominant negative inhibition of myosin activity. We found that interference with the functions of three class VIII myosins and two class XI myosins significantly reduced the local and long-distance transport of the virus. We further determined that the inactivation of myosins XI-2 and XI-K affected the structure and dynamic behavior of the ER leading to aggregation of the viral movement protein (MP) and to a delay in the MP accumulation in plasmodesmata (PD). The inactivation of myosin XI-2 but not of myosin XI-K affected the localization pattern of the 126k replicase subunit and the level of TMV accumulation. The inhibition of myosins VIII-1, VIII-2 and VIII-B abolished MP localization to PD and caused its retention at the plasma membrane. These results suggest that class XI myosins contribute to the viral propagation and intracellular trafficking, whereas myosins VIII are specifically required for the MP targeting to and virus movement through the PD. Thus, TMV appears to recruit distinct myosins for different steps in the cell-to-cell spread of the infection.