Joshua J. Blakeslee

ABCB19/PGP19 stabilises PIN1 in membrane microdomains in Arabidopsis

Auxin transport is mediated at the cellular level by three independent mechanisms that are characterised by the PIN-formed (PIN), P-glycoprotein (ABCB/PGP) and AUX/LAX transport proteins. The PIN and ABCB transport proteins, best represented by PIN1 and ABCB19 (PGP19), have been shown to coordinately regulate auxin efflux. When PIN1 and ABCB19 coincide on the plasma membrane, their interaction enhances the rate and specificity of auxin efflux and the dynamic cycling of PIN1 is reduced. However, ABCB19 function is not regulated by the dynamic cellular trafficking mechanisms that regulate PIN1 in apical tissues, as localisation of ABCB19 on the plasma membrane was not inhibited by short-term treatments with latrunculin B, oryzalin, brefeldin A (BFA) or wortmannin--all of which have been shown to alter PIN1 and/or PIN2 plasma membrane localisation. When taken up by endocytosis, the styryl dye FM4-64 labels diffuse rather than punctuate intracellular bodies in abcb19 (pgp19), and some aggregations of PIN1 induced by short-term BFA treatment did not disperse after BFA washout in abcb19. Although the subcellular localisations of ABCB19 and PIN1 in the reciprocal mutant backgrounds were like those in wild type, PIN1 plasma membrane localisation in abcb19 roots was more easily perturbed by the detergent Triton X-100, but not other non-ionic detergents. ABCB19 is stably associated with sterol/sphingolipid-enriched membrane fractions containing BIG/TIR3 and partitions into Triton X-100 detergent-resistant membrane (DRM) fractions. In the wild type, PIN1 was also present in DRMs, but was less abundant in abcb19 DRMs. These observations suggested a rationale for the observed lack of auxin transport activity when PIN1 is expressed in a non-plant heterologous system. PIN1 was therefore expressed in Schizosaccharomyces pombe, which has plant-like sterol-enriched microdomains, and catalysed auxin transport in these cells. These data suggest that ABCB19 stabilises PIN1 localisation at the plasma membrane in discrete cellular subdomains where PIN1 and ABCB19 expression overlaps.

Interactions among PIN-FORMED and P-Glycoprotein Auxin Transporters in Arabidopsis

Directional transport of the phytohormone auxin is established primarily at the point of cellular efflux and is required for the establishment and maintenance of plant polarity. Studies in whole plants and heterologous systems indicate that PIN-FORMED (PIN) and P-glycoprotein (PGP) transport proteins mediate the cellular efflux of natural and synthetic auxins. However, aromatic anion transport resulting from PGP and PIN expression in nonplant systems was also found to lack the high level of substrate specificity seen in planta. Furthermore, previous reports that PGP19 stabilizes PIN1 on the plasma membrane suggested that PIN–PGP interactions might regulate polar auxin efflux. Here, we show that PGP1 and PGP19 colocalized with PIN1 in the shoot apex in Arabidopsis thaliana and with PIN1 and PIN2 in root tissues. Specific PGP–PIN interactions were seen in yeast two-hybrid and coimmunoprecipitation assays. PIN–PGP interactions appeared to enhance transport activity and, to a greater extent, substrate/inhibitor specificities when coexpressed in heterologous systems. By contrast, no interactions between PGPs and the AUXIN1 influx carrier were observed. Phenotypes of pin and pgp mutants suggest discrete functional roles in auxin transport, but pin pgp mutants exhibited phenotypes that are both additive and synergistic. These results suggest that PINs and PGPs characterize coordinated, independent auxin transport mechanisms but also function interactively in a tissue-specific manner.

Variation in Expression and Protein Localization of the PIN Family of Auxin Efflux Facilitator Proteins in Flavonoid Mutants with Altered Auxin Transport in Arabidopsis thaliana

Aglycone flavonols are thought to modulate auxin transport in Arabidopsis thaliana via an as yet undefined mechanism. Biochemical studies suggest that flavonoids interact with regulatory proteins rather than directly with the PIN auxin efflux facilitator proteins. Auxin transport is enhanced in the absence of flavonoids (transparent testa4 [tt4]) and reduced in the presence of excess flavonols (tt7 and tt3). Steady state PIN mRNA levels in roots inversely correlate with auxin movement in tt mutants. PIN gene transcription and protein localization in flavonoid-deficient mutants appear to be modulated by developmental cues and are auxin responsive. Modulation of PIN gene expression and protein distribution by localized auxin accumulations occurs in the wild type as well. Flavonoids inhibit auxin transport primarily at the shoot apex and root tip and appear to modulate vesicular cycling of PIN1 at the root tip. In some auxin-accumulating tissues, flavonoid increases and changes in flavonoid speciation are subsequent to auxin accumulation.

PGP4, an ATP Binding Cassette P-Glycoprotein, Catalyzes Auxin Transport in Arabidopsis thaliana Roots

Members of the ABC (for ATP binding cassette) superfamily of integral membrane transporters function in cellular detoxification, cell-to-cell signaling, and channel regulation. More recently, members of the multidrug resistance P-glycoprotein (MDR/PGP) subfamily of ABC transporters have been shown to function in the transport of the phytohormone auxin in both monocots and dicots. Here, we report that the Arabidopsis thaliana MDR/PGP PGP4 functions in the basipetal redirection of auxin from the root tip. Reporter gene studies showed that PGP4 was strongly expressed in root cap and epidermal cells. PGP4 exhibits apolar plasma membrane localization in the root cap and polar localization in tissues above. Root gravitropic bending and elongation as well as lateral root formation were reduced in pgp4 mutants compared with the wild type. pgp4 exhibited reduced basipetal auxin transport in roots and a small decrease in shoot-to-root transport consistent with a partial loss of the redirective auxin sink in the root. Seedlings overexpressing PGP4 exhibited increased shoot-to-root auxin transport. Heterologous expression of PGP4 in mammalian cells resulted in 1-N-naphthylthalamic acid–reversible net uptake of [3H]indole-3-acetic acid. These results indicate that PGP4 functions primarily in the uptake of redirected or newly synthesized auxin in epidermal root cells.

Differential Effects of Sucrose and Auxin on Localized Phosphate Deficiency-Induced Modulation of Different Traits of Root System Architecture in Arabidopsis

Phosphorus, one of the essential elements for plants, is often a limiting nutrient in soils. Low phosphate (Pi) availability induces sugar-dependent systemic expression of genes and modulates the root system architecture (RSA). Here, we present the differential effects of sucrose (Suc) and auxin on the Pi deficiency responses of the primary and lateral roots of Arabidopsis (Arabidopsis thaliana). Inhibition of primary root growth and loss of meristematic activity were evident in seedlings grown under Pi deficiency with or without Suc. Although auxin supplementation also inhibited primary root growth, loss of meristematic activity was observed specifically under Pi deficiency with or without Suc. The results suggested that Suc and auxin do not influence the mechanism involved in localized Pi sensing that regulates growth of the primary root and therefore delineates it from sugar-dependent systemic Pi starvation responses. However, the interaction between Pi and Suc was evident on the development of the lateral roots and root hairs in the seedlings grown under varying levels of Pi and Suc. Although the Pi+ Suc− condition suppressed lateral root development, induction of few laterals under the Pi− Suc− condition point to increased sensitivity of the roots to auxin during Pi deprivation. This was supported by expression analyses of DR5∷uidA, root basipetal transport assay of auxin, and RSA of the pgp19 mutant exhibiting reduced auxin transport. A significant increase in the number of lateral roots under the Pi− Suc− condition in the chalcone synthase mutant (tt4-2) indicated a potential role for flavonoids in auxin-mediated Pi deficiency-induced modulation of RSA. The study thus demonstrated differential roles of Suc and auxin in the developmental responses of ontogenetically distinct root traits during Pi deprivation. In addition, lack of cross talk between local and systemic Pi sensing as revealed by the seedlings grown under either the Pi− Suc− condition or in the heterogenous Pi environment highlighted the coexistence of Suc-independent and Suc-dependent regulatory mechanisms that constitute Pi starvation responses.

Seven things we think we know about auxin transport

Polar transport of the phytohormone auxin and the establishment of localized auxin maxima regulate embryonic development, stem cell maintenance, root and shoot architecture, and tropic growth responses. The past decade has been marked by dramatic progress in efforts to elucidate the complex mechanisms by which auxin transport regulates plant growth. As the understanding of auxin transport regulation has been increasingly elaborated, it has become clear that this process is involved in almost all plant growth and environmental responses in some way. However, we still lack information about some basic aspects of this fundamental regulatory mechanism. In this review, we present what we know (or what we think we know) and what we do not know about seven auxin-regulated processes. We discuss the role of auxin transport in gravitropism in primary and lateral roots, phototropism, shoot branching, leaf expansion, and venation. We also discuss the auxin reflux/fountain model at the root tip, flavonoid modulation of auxin transport processes, and outstanding aspects of post-translational regulation of auxin transporters. This discussion is not meant to be exhaustive, but highlights areas in which generally held assumptions require more substantive validation.

Immunophilin-like TWISTED DWARF1 Modulates Auxin Efflux Activities of Arabidopsis P-glycoproteins

The immunophilin-like protein TWISTED DWARF1 (TWD1/FKBP42) has been shown to physically interact with the multidrug resistance/P-glycoprotein (PGP) ATP-binding cassette transporters PGP1 and PGP19 (MDR1). Overlapping phenotypes of pgp1/pgp19 and twd1 mutant plants suggested a positive regulatory role of TWD1 in PGP-mediated export of the plant hormone auxin, which controls plant development. Here, we provide evidence at the cellular and plant levels that TWD1 controls PGP-mediated auxin transport. twd1 and pgp1/pgp19 cells showed greatly reduced export of the native auxin indole-3-acetic acid (IAA). Constitutive overexpression of PGP1 and PGP19, but not TWD1, enhanced auxin export. Coexpression of TWD1 and PGP1 in yeast and mammalian cells verified the specificity of the regulatory effect. Employing an IAA-specific microelectrode demonstrated that IAA influx in the root elongation zone was perturbed and apically shifted in pgp1/pgp19 and twd1 roots. Mature roots of pgp1/pgp19 and twd1 plants revealed elevated levels of free IAA, which seemed to account for agravitropic root behavior. Our data suggest a novel mode of PGP regulation via FK506-binding protein-like immunophilins, implicating possible alternative strategies to overcome multidrug resistance.

The VTI Family of SNARE Proteins Is Necessary for Plant Viability and Mediates Different Protein Transport Pathways

The Arabidopsis genome contains a family of v-SNAREs: VTI11, VTI12, and VTI13. Only VTI11 and VTI12 are expressed at appreciable levels. Although these two proteins are 60% identical, they complement different transport pathways when expressed in the yeast vti1 mutant. VTI11 was identified recently as the mutated gene in the shoot gravitropic mutant zig. Here, we show that the vti11 zig mutant has defects in vascular patterning and auxin transport. An Arabidopsis T-DNA insertion mutant, vti12, had a normal phenotype under nutrient-rich growth conditions. However, under nutrient-poor conditions, vti12 showed an accelerated senescence phenotype, suggesting that VTI12 may play a role in the plant autophagy pathway. VTI11 and VTI12 also were able to substitute for each other in their respective SNARE complexes, and a double-mutant cross between zig and vti12 was embryo lethal. These results suggest that some VTI1 protein was necessary for plant viability and that the two proteins were partially functionally redundant.

Relocalization of the PIN1 auxin efflux facilitator plays a role in phototropic responses

Recently, we reported that the basal localization of the PIN1 auxin efflux facilitator protein is disrupted in hypocotyls of Arabidopsis mdr ( pgp ) mutants grown in the dark or unidirectional light ([Noh et al., 2003][1]). Molecular genetic and physiological evidence indicates that PIN1 is required

Reduction of Benzenoid Synthesis in Petunia Flowers Reveals Multiple Pathways to Benzoic Acid and Enhancement in Auxin Transport

In plants, benzoic acid (BA) is believed to be synthesized from Phe through shortening of the propyl side chain by two carbons. It is hypothesized that this chain shortening occurs via either a β-oxidative or non-β-oxidative pathway. Previous in vivo isotope labeling and metabolic flux analysis of the benzenoid network in petunia (Petunia hybrida) flowers revealed that both pathways yield benzenoid compounds and that benzylbenzoate is an intermediate between l-Phe and BA. To test this hypothesis, we generated transgenic petunia plants in which the expression of BPBT, the gene encoding the enzyme that uses benzoyl-CoA and benzyl alcohol to make benzylbenzoate, was reduced or eliminated. Elimination of benzylbenzoate formation decreased the endogenous pool of BA and methylbenzoate emission but increased emission of benzyl alcohol and benzylaldehyde, confirming the contribution of benzylbenzoate to BA formation. Labeling experiments with 2H5-Phe revealed a dilution of isotopic abundance in most measured compounds in the dark, suggesting an alternative pathway from a precursor other than Phe, possibly phenylpyruvate. Suppression of BPBT activity also affected the overall morphology of petunia plants, resulting in larger flowers and leaves, thicker stems, and longer internodes, which was consistent with the increased auxin transport in transgenic plants. This suggests that BPBT is involved in metabolic processes in vegetative tissues as well.