Bangjun Wang

A novel putative auxin carrier family regulates intracellular auxin homeostasis in plants

The phytohormone auxin acts as a prominent signal, providing, by its local accumulation or depletion in selected cells, a spatial and temporal reference for changes in the developmental program. The distribution of auxin depends on both auxin metabolism (biosynthesis, conjugation and degradation) and cellular auxin transport. We identified in silico a novel putative auxin transport facilitator family, called PIN-LIKES (PILS). Here we illustrate that PILS proteins are required for auxin-dependent regulation of plant growth by determining the cellular sensitivity to auxin. PILS proteins regulate intracellular auxin accumulation at the endoplasmic reticulum and thus auxin availability for nuclear auxin signalling. PILS activity affects the level of endogenous auxin indole-3-acetic acid (IAA), presumably via intracellular accumulation and metabolism. Our findings reveal that the transport machinery to compartmentalize auxin within the cell is of an unexpected molecular complexity and demonstrate this compartmentalization to be functionally important for a number of developmental processes.

ER-localized auxin transporter PIN8 regulates auxin homeostasis and male gametophyte development in Arabidopsis

Auxin is a key coordinative signal required for many aspects of plant development and its levels are controlled by auxin metabolism and intercellular auxin transport. Here we find that a member of PIN auxin transporter family, PIN8 is expressed in male gametophyte of Arabidopsis thaliana and has a crucial role in pollen development and functionality. Ectopic expression in sporophytic tissues establishes a role of PIN8 in regulating auxin homoeostasis and metabolism. PIN8 co-localizes with PIN5 to the endoplasmic reticulum (ER) where it acts as an auxin transporter. Genetic analyses reveal an antagonistic action of PIN5 and PIN8 in the regulation of intracellular auxin homoeostasis and gametophyte as well as sporophyte development. Our results reveal a role of the auxin transport in male gametophyte development in which the distinct actions of ER-localized PIN transporters regulate cellular auxin homoeostasis and maintain the auxin levels optimal for pollen development and pollen tube growth.

Export of salicylic acid from the chloroplast requires the multidrug and toxin extrusion-like transporter EDS5.

The synthesis of the immune signal salicylic acid is abolished by a mutation in a hitherto unknown transporter protein. This article describes the transporter localization at the chloroplast and its function in the export of salicylic acid from the chloroplast. Salicylic acid (SA) is central for the defense of plants to pathogens and abiotic stress. SA is synthesized in chloroplasts from chorismic acid by an isochorismate synthase (ICS1); SA biosynthesis is negatively regulated by autoinhibitory feedback at ICS1. Genetic studies indicated that the multidrug and toxin extrusion transporter ENHANCED DISEASE SUSCEPTIBILITY5 (EDS5) of Arabidopsis (Arabidopsis thaliana) is necessary for SA accumulation after biotic and abiotic stress, but so far it is not understood how EDS5 controls the biosynthesis of SA. Here, we show that EDS5 colocalizes with a marker of the chloroplast envelope and that EDS5 functions as a multidrug and toxin extrusion-like transporter in the export of SA from the chloroplast to the cytoplasm in Arabidopsis, where it controls the innate immune response. The location at the chloroplast envelope supports a model of the effect of EDS5 on SA biosynthesis: in the eds5 mutant, stress-induced SA is trapped in the chloroplast and inhibits its own accumulation by autoinhibitory feedback.

Regulation of ABCB1/PGP1‐catalysed auxin transport by linker phosphorylation

Polar transport of the plant hormone auxin is controlled by PIN‐ and ABCB/PGP‐efflux catalysts. PIN polarity is regulated by the AGC protein kinase, PINOID (PID), while ABCB activity was shown to be dependent on interaction with the FKBP42, TWISTED DWARF1 (TWD1). Using co‐immunoprecipitation (co‐IP) and shotgun LC–MS/MS analysis, we identified PID as a valid partner in the interaction with TWD1. In‐vitro and yeast expression analyses indicated that PID specifically modulates ABCB1‐mediated auxin efflux in an action that is dependent on its kinase activity and that is reverted by quercetin binding and thus inhibition of PID autophosphorylation. Triple ABCB1/PID/TWD1 co‐transfection in tobacco revealed that PID enhances ABCB1‐mediated auxin efflux but blocks ABCB1 in the presence of TWD1. Phospho‐proteomic analyses identified S634 as a key residue of the regulatory ABCB1 linker and a very likely target of PID phosphorylation that determines both transporter drug binding and activity. In summary, we provide evidence that PID phosphorylation has a dual, counter‐active impact on ABCB1 activity that is coordinated by TWD1–PID interaction.

Arabidopsis ABCB21 is a facultative auxin importer/exporter regulated by cytoplasmic auxin concentration.

The phytohormone auxin is critical for plant growth and many developmental processes. Members of the P-glycoprotein (PGP/ABCB) subfamily of ATP-binding cassette (ABC) transporters have been shown to function in the polar movement of auxin by transporting auxin over the plasma membrane in both monocots and dicots. Here, we characterize a new Arabidopsis member of the ABCB subfamily, ABCB21/PGP21, a close homolog of ABCB4, for which conflicting transport directionalities have been reported. ABCB21 is strongly expressed in the abaxial side of cotyledons and in junctions of lateral organs in the aerial part, whereas in roots it is specifically expressed in pericycle cells. Membrane fractionation by sucrose density gradient centrifugation followed by Western blot showed that ABCB21 is a plasma membrane-localized ABC transporter. A transport assay with Arabidopsis protoplasts suggested that ABCB21 was involved in IAA transport in an outward direction, while naphthalene acetic acid (NAA) was a less preferable substrate for ABCB21. Further functional analysis of ABCB21 using yeast import and export assays showed that ABCB21 mediates the 1-N-naphthylphthalamic acid (NPA)-sensitive translocation of auxin in an inward direction when the cytoplasmic IAA concentration is low, whereas this transporter mediates outward transport under high internal IAA. An increase in the cytoplasmic IAA concentration by pre-loading of IAA into yeast cells abolished the IAA uptake activity by ABCB21 as well as ABCB4. These findings suggest that ABCB21 functions as a facultative importer/exporter controlling auxin concentrations in plant cells.

Arabidopsis TWISTED DWARF1 Functionally Interacts with Auxin Exporter ABCB1 on the Root Plasma Membrane

The export of auxin by ABCB-type auxin transporters is essential for proper plant development and is regulated by TWISTED DWARF1. This work shows that in addition to the endoplasmic reticulum, TWISTED DWARF1 is also located at lateral plasma membrane subdomains where it colocalizes and interacts with ABCB1; the data support a model in which TWISTED DWARF1 promotes lateral ABCB1-mediated auxin efflux at the plasma membrane. Plant architecture is influenced by the polar, cell-to-cell transport of auxin that is primarily provided and regulated by plasma membrane efflux catalysts of the PIN-FORMED and B family of ABC transporter (ABCB) classes. The latter were shown to require the functionality of the FK506 binding protein42 TWISTED DWARF1 (TWD1), although underlying mechanisms are unclear. By genetic manipulation of TWD1 expression, we show here that TWD1 affects shootward root auxin reflux and, thus, downstream developmental traits, such as epidermal twisting and gravitropism of the root. Using immunological assays, we demonstrate a predominant lateral, mainly outward-facing, plasma membrane location for TWD1 in the root epidermis characterized by the lateral marker ABC transporter G36/PLEIOTROPIC DRUG-RESISTANCE8/PENETRATION3. At these epidermal plasma membrane domains, TWD1 colocalizes with nonpolar ABCB1. In planta bioluminescence resonance energy transfer analysis was used to verify specific ABC transporter B1 (ABCB1)–TWD1 interaction. Our data support a model in which TWD1 promotes lateral ABCB-mediated auxin efflux via protein–protein interaction at the plasma membrane, minimizing reflux from the root apoplast into the cytoplasm.

Auxin transport during root gravitropism: transporters and techniques

Root gravitropism is a complex, plant-specific process allowing roots to grow downward into the soil. Polar auxin transport and redistribution are essential for root gravitropism. Here we summarise our current understanding of underlying molecular mechanisms and involved transporters that establish, maintain and redirect intercellular auxin gradients as the driving force for root gravitropism. We evaluate the genetic, biochemical and cell biological approaches presently used for the analysis of auxin redistribution and the quantification of auxin fluxes. Finally, we also discuss new tools that provide a higher spatial or temporal resolution and our technical needs for future gravitropism studies.

The cyclophilin A DIAGEOTROPICA gene affects auxin transport in both root and shoot to control lateral root formation

Cyclophilin A is a conserved peptidyl-prolyl cis-trans isomerase (PPIase) best known as the cellular receptor of the immunosuppressant cyclosporine A. Despite significant effort, evidence of developmental functions of cyclophilin A in non-plant systems has remained obscure. Mutations in a tomato (Solanum lycopersicum) cyclophilin A ortholog, DIAGEOTROPICA (DGT), have been shown to abolish the organogenesis of lateral roots; however, a mechanistic explanation of the phenotype is lacking. Here, we show that the dgt mutant lacks auxin maxima relevant to priming and specification of lateral root founder cells. DGT is expressed in shoot and root, and localizes to both the nucleus and cytoplasm during lateral root organogenesis. Mutation of ENTIRE/IAA9, a member of the auxin-responsive Aux/IAA protein family of transcriptional repressors, partially restores the inability of dgt to initiate lateral root primordia but not the primordia outgrowth. By comparison, grafting of a wild-type scion restores the process of lateral root formation, consistent with participation of a mobile signal. Antibodies do not detect movement of the DGT protein into the dgt rootstock; however, experiments with radiolabeled auxin and an auxin-specific microelectrode demonstrate abnormal auxin fluxes. Functional studies of DGT in heterologous yeast and tobacco-leaf auxin-transport systems demonstrate that DGT negatively regulates PIN-FORMED (PIN) auxin efflux transporters by affecting their plasma membrane localization. Studies in tomato support complex effects of the dgt mutation on PIN expression level, expression domain and plasma membrane localization. Our data demonstrate that DGT regulates auxin transport in lateral root formation. Summary: DIAGEOTROPICA modulates the localization of PIN proteins and is required for proper polar auxin transport, highlighting a new developmental role for this cyclophilin.

The AGC kinase, PINOID, blocks interactive ABCB/PIN auxin transport

Plant growth and development is determined by intracellular and intercellular auxin gradients that are controlled at first hand by auxin efflux catalysts of the ABCB/PGP and PIN families. ABCB transport activity was shown to be counter-actively regulated by protein phosphorylation by the AGC protein kinase, PINOID (PID), that is coordinated by interaction with the immunophilin-like FKBP42, TWISTED DWARF1 (TWD1). In contrast, PID was shown to determine PIN polarity, however, the direct impact of PID on PIN activity has yet not been tested. Co-expression in yeast indicates that PID had no effect on PIN1,2 alone but specifically inhibits interactive ABCB1-PIN1/PIN2 auxin efflux in an action that is dependent on its kinase activity. PIN1-PID co-transfection in N. benthamiana revealed that PID blocks PIN1-mediated auxin efflux without changing PIN1 location. In summary, these data provide evidence that PID phosphorylation does not only determine PIN polarity but also has a direct impact on transport activity of the activity of the binary PIN-ABCB1 complex.

Expression of TWISTED DWARF1 lacking its in‐plane membrane anchor leads to increased cell elongation and hypermorphic growth

Plant growth is achieved predominantly by cellular elongation, which is thought to be controlled on several levels by apoplastic auxin. Auxin export into the apoplast is achieved by plasma membrane efflux catalysts of the PIN-FORMED (PIN) and ATP-binding cassette protein subfamily B/phosphor-glycoprotein (ABCB/PGP) classes; the latter were shown to depend on interaction with the FKBP42, TWISTED DWARF1 (TWD1). Here by using a transgenic approach in combination with phenotypical, biochemical and cell biological analyses we demonstrate the importance of a putative C-terminal in-plane membrane anchor of TWD1 in the regulation of ABCB-mediated auxin transport. In contrast with dwarfed twd1 loss-of-function alleles, TWD1 gain-of-function lines that lack a putative in-plane membrane anchor (HA-TWD1-Ct ) show hypermorphic plant architecture, characterized by enhanced stem length and leaf surface but reduced shoot branching. Greater hypocotyl length is the result of enhanced cell elongation that correlates with reduced polar auxin transport capacity for HA-TWD1-Ct . As a consequence, HA-TWD1-Ct displays higher hypocotyl auxin accumulation, which is shown to result in elevated auxin-induced cell elongation rates. Our data highlight the importance of C-terminal membrane anchoring for TWD1 action, which is required for specific regulation of ABCB-mediated auxin transport. These data support a model in which TWD1 controls lateral ABCB1-mediated export into the apoplast, which is required for auxin-mediated cell elongation.