Thermal infrared directs host-seeking behaviour in Aedes aegypti mosquitoes.

Mosquito-borne diseases affect hundreds of millions of people annually and disproportionately impact the developing world1,2. One mosquito species, Aedes aegypti, is a primary vector of viruses that cause dengue, yellow fever and Zika. The attraction of Ae. aegypti female mosquitos to humans requires integrating multiple cues, including CO2 from breath, organic odours from skin and visual cues, all sensed at mid and long ranges, and other cues sensed at very close range3-6. Here we identify a cue that Ae. aegypti use as part of their sensory arsenal to find humans. We demonstrate that Ae. aegypti sense the infrared (IR) radiation emanating from their targets and use this information in combination with other cues for highly effective mid-range navigation. Detection of thermal IR requires the heat-activated channel TRPA1, which is expressed in neurons at the tip of the antenna. Two opsins are co-expressed with TRPA1 in these neurons and promote the detection of lower IR intensities. We propose that radiant energy causes local heating at the end of the antenna, thereby activating temperature-sensitive receptors in thermosensory neurons. The realization that thermal IR radiation is an outstanding mid-range directional cue expands our understanding as to how mosquitoes are exquisitely effective in locating hosts.

Endocrine modulation of primary chemosensory neurons regulates Drosophila courtship behavior.

The decision to engage in courtship depends on external cues from potential mates and internal cues related to maturation, health, and experience. Hormones allow for coordinated conveyance of such information to peripheral tissues. Here, we show Ecdysis-Triggering Hormone (ETH) is critical for courtship inhibition after completion of copulation in Drosophila melanogaster. ETH deficiency relieves post-copulation courtship inhibition (PCCI) and increases male-male courtship. ETH appears to modulate perception and attractiveness of potential mates by direct action on primary chemosensory neurons. Knockdown of ETH receptor (ETHR) expression in GR32A-expressing neurons leads to reduced ligand sensitivity and elevated male-male courtship. We find OR67D also is critical for normal levels of PCCI after mating. ETHR knockdown in OR67D-expressing neurons or GR32A-expressing neurons relieves PCCI. Finally, ETHR silencing in the corpus allatum (CA), the sole source of juvenile hormone, also relieves PCCI; treatment with the juvenile hormone analog methoprene partially restores normal post-mating behavior. We find that ETH, a stress-sensitive reproductive hormone, appears to coordinate multiple sensory modalities to guide Drosophila male courtship behaviors, especially after mating.

Calcium-dependent protein kinase 29 modulates PIN-FORMED polarity and Arabidopsis development via its own phosphorylation code.

PIN-FORMED (PIN)-mediated polar auxin transport (PAT) is involved in key developmental processes in plants. Various internal and external cues influence plant development via the modulation of intracellular PIN polarity and, thus, the direction of PAT, but the mechanisms underlying these processes remain largely unknown. PIN proteins harbor a hydrophilic loop (HL) that has important regulatory functions; here, we used the HL as bait in protein pulldown screening for modulators of intracellular PIN trafficking in Arabidopsis thaliana. Calcium-dependent protein kinase 29 (CPK29), a Ca2+-dependent protein kinase, was identified and shown to phosphorylate specific target residues on the PIN-HL that were not phosphorylated by other kinases. Furthermore, loss of CPK29 or mutations of the phospho-target residues in PIN-HLs significantly compromised intracellular PIN trafficking and polarity, causing defects in PIN-mediated auxin redistribution and biological processes such as lateral root formation, root twisting, hypocotyl gravitropism, phyllotaxis, and reproductive development. These findings indicate that CPK29 directly interprets Ca2+ signals from internal and external triggers, resulting in the modulation of PIN trafficking and auxin responses.

Internal Structure of Incipient Soot from Acetylene Pyrolysis Obtained via Molecular Dynamics Simulations.

A series of reactive molecular dynamics simulations is used to study the internal structure of incipient soot particles obtained from acetylene pyrolysis. The simulations were performed using the ReaxFF potential at four different temperatures. The resulting soot particles are cataloged and analyzed to obtain statistics of their mass, volume, density, C/H ratio, number of cyclic structures, and other features. A total of 3324 incipient soot particles were analyzed in this study. Based on their structural characteristics, the incipient soot particles are classified into two classes, termed type 1 and type 2 incipient soot particles in this work. The radial distribution of density, cyclic (5-, 6-, or 7-member rings) structures, and C/H ratio inside the particles revealed a clear difference in the internal structure between type 1 and type 2 particles. These classes were further found to be well represented by the size of the particles, with smaller particles in type 1 and larger particles in type 2. The radial distributions of ring structures, density, and the C/H ratio indicated the presence of a dense core region in type 2 particles. In contrast, no clear evidence of the presence of a core was found in type 1 particles. In type 2 incipient soot particles, the boundary between the core and shell was found to be around 50-60% of the particle's radius of gyration.

Requirement for an Otopetrin-like protein for acid taste in Drosophila.

Receptors for bitter, sugar, and other tastes have been identified in the fruit fly Drosophila melanogaster, while a broadly tuned receptor for the taste of acid has been elusive. Previous work showed that such a receptor was unlikely to be encoded by a gene within one of the two major families of taste receptors in Drosophila, the "gustatory receptors" and "ionotropic receptors." Here, to identify the acid taste receptor, we tested the contributions of genes encoding proteins distantly related to the mammalian Otopertrin1 (OTOP1) proton channel that functions as a sour receptor in mice. RNA interference (RNAi) knockdown or mutation by CRISPR/Cas9 of one of the genes, Otopetrin-Like A (OtopLA), but not of the others (OtopLB or OtopLC) severely impaired the behavioral rejection to a sweet solution laced with high levels of HCl or carboxylic acids and greatly reduced acid-induced action potentials measured from taste hairs. An isoform of OtopLA that we isolated from the proboscis was sufficient to restore behavioral sensitivity and acid-induced action potential firing in OtopLA mutant flies. At lower concentrations, HCl was attractive to the flies, and this attraction was abolished in the OtopLA mutant. Cell type-specific rescue experiments showed that OtopLA functions in distinct subsets of gustatory receptor neurons for repulsion and attraction to high and low levels of protons, respectively. This work highlights a functional conservation of a sensory receptor in flies and mammals and shows that the same receptor can function in both appetitive and repulsive behaviors.

FRA1 Kinesin Modulates the Lateral Stability of Cortical Microtubules through Cellulose Synthase-Microtubule Uncoupling Proteins.

Cell wall assembly requires harmonized deposition of cellulose and matrix polysaccharides. Cortical microtubules orient the deposition of cellulose by guiding the trajectory of cellulose synthase complexes. Vesicles containing matrix polysaccharides are thought to be transported by the FRAGILE FIBER1 (FRA1) kinesin to facilitate their secretion along cortical microtubules. The cortical microtubule cytoskeleton thus may provide a platform to coordinate the delivery of cellulose and matrix polysaccharides, but the underlying molecular mechanisms remain unknown. Here, we show that the tail region of the Arabidopsis (Arabidopsis thaliana) FRA1 kinesin physically interacts with cellulose synthase-microtubule uncoupling (CMU) proteins that are important for the microtubule-dependent guidance of cellulose synthase complexes. Interaction with CMUs did not affect microtubule binding or motility of the FRA1 kinesin but differentially affected the protein levels and microtubule localization of CMU1 and CMU2, thus regulating the lateral stability of cortical microtubules. Phosphorylation of the FRA1 tail region inhibited binding to CMUs and consequently reversed the extent of cortical microtubule decoration by CMU1 and CMU2. Genetic experiments demonstrated the significance of this interaction to the growth and reproduction of Arabidopsis plants. We propose that modulation of CMU protein levels and microtubule localization by FRA1 provides a mechanism that stabilizes the sites of deposition of both cellulose and matrix polysaccharides.

An inhibitory mechanism for suppressing high salt intake in Drosophila.

High concentrations of dietary salt are harmful to health. Like most animals, Drosophila melanogaster are attracted to foods that have low concentrations of salt, but show strong taste avoidance of high salt foods. Salt in known on multiple classes of taste neurons, activating Gr64f sweet-sensing neurons that drive food acceptance and 2 others (Gr66a bitter and Ppk23 high salt) that drive food rejection. Here we find that NaCl elicits a bimodal dose-dependent response in Gr64f taste neurons, which show high activity with low salt and depressed activity with high salt. High salt also inhibits the sugar response of Gr64f neurons, and this action is independent of the neuron's taste response to salt. Consistent with the electrophysiological analysis, feeding suppression in the presence of salt correlates with inhibition of Gr64f neuron activity, and remains if high salt taste neurons are genetically silenced. Other salts such as Na2SO4, KCl, MgSO4, CaCl2, and FeCl3 act on sugar response and feeding behavior in the same way. A comparison of the effects of various salts suggests that inhibition is dictated by the cationic moiety rather than the anionic component of the salt. Notably, high salt-dependent inhibition is not observed in Gr66a neurons-response to a canonical bitter tastant, denatonium, is not altered by high salt. Overall, this study characterizes a mechanism in appetitive Gr64f neurons that can deter ingestion of potentially harmful salts.

Dietary Macronutrient Imbalances Lead to Compensatory Changes in Peripheral Taste via Independent Signaling Pathways.

Food choice, in animals, has been known to change with internal nutritional state and also with variable dietary conditions. To better characterize mechanisms of diet-induced plasticity of food preference in Drosophila melanogaster, we synthesized diets with macronutrient imbalances and examined how food choice and taste sensitivity were modified in flies that fed on these diets. We found that dietary macronutrient imbalances caused compensatory behavioral shifts in both sexes to increase preference for the macronutrient that was scant in the food source, and simultaneously reduce preference for the macronutrient that was enriched. Further analysis with females revealed analogous changes in sweet taste responses in labellar neurons, with increased sensitivity on sugar-reduced diet and decreased sensitivity on sugar-enriched diet. Interestingly, we found differences in the onset of changes in taste sensitivity and behavior, which occur over 1-4 d, in response to dietary sugar reduction or enrichment. To investigate molecular mechanisms responsible for diet-induced taste modulation, we used candidate gene and transcriptome analyses. Our results indicate that signaling via Dop2R is involved in increasing cellular and behavioral sensitivity to sugar as well as in decreasing behavioral sensitivity to amino acids on dietary sugar reduction. On the other hand, cellular and behavioral sensitivity to sugar relies on dilp5 and a decrease in sugar preference following dietary sugar abundance was correlated with downregulation of dilp5 Together, our results suggest that feeding preference for sugar and amino acid can be modulated independently to facilitate food choice that accounts for prior dietary experience.SIGNIFICANCE STATEMENT Animals adjust their feeding preferences based on prior dietary experiences. Here, we find that upon dietary macronutrient deprivation, flies undergo compensatory changes in food preference. The altered preference correlates with changes in peripheral taste sensitivity. While Dop2R mediates changes following dietary sugar reduction, downregulation of dilp5 is associated with changes caused by a sugar-enriched diet. This study contributes to a better understanding of neurophysiological plasticity of the taste system in flies, and its role in facilitating adjustment of foraging behavior based on nutritional requirements.

The Arabidopsis kinesin-4, FRA1, requires a high level of processive motility to function correctly.

Processivity is important for kinesins that mediate intracellular transport. Structure-function analyses of N-terminal kinesins (i.e. kinesins comprising their motor domains at the N-terminus) have identified several non-motor regions that affect processivity in vitro However, whether these structural elements affect kinesin processivity and function in vivo is not known. Here, we used an Arabidopsis thaliana kinesin-4, called Fragile Fiber 1 (FRA1, also known as KIN4A), which is thought to mediate vesicle transport, to test whether mutations that alter processivity in vitro lead to similar changes in behavior in vivo and whether processivity is important for the function of FRA1. We generated several FRA1 mutants that differed in their 'run lengths' in vitro and then transformed them into the fra1-5 mutant for complementation and in vivo motility analyses. Our data show that the behavior of processivity mutants in vivo can differ dramatically from in vitro properties, underscoring the need to extend structure-function analyses of kinesins in vivo In addition, we found that a high density of processive motility is necessary for the physiological function of FRA1.

Functional Analysis of the Hydrophilic Loop in Intracellular Trafficking of Arabidopsis PIN-FORMED Proteins.

Different PIN-FORMED proteins (PINs) contribute to intercellular and intracellular auxin transport, depending on their distinctive subcellular localizations. Arabidopsis thaliana PINs with a long hydrophilic loop (HL) (PIN1 to PIN4 and PIN7; long PINs) localize predominantly to the plasma membrane (PM), whereas short PINs (PIN5 and PIN8) localize predominantly to internal compartments. However, the subcellular localization of the short PINs has been observed mostly for PINs ectopically expressed in different cell types, and the role of the HL in PIN trafficking remains unclear. Here, we tested whether a long PIN-HL can provide its original molecular cues to a short PIN by transplanting the HL. The transplanted long PIN2-HL was sufficient for phosphorylation and PM trafficking of the chimeric PIN5:PIN2-HL but failed to provide the characteristic polarity of PIN2. Unlike previous observations, PIN5 showed clear PM localization in diverse cell types where PIN5 is natively or ectopically expressed and even polar PM localization in one cell type. Furthermore, in the root epidermis, the subcellular localization of PIN5 switched from PM to internal compartments according to the developmental stage. Our results suggest that the long PIN-HL is partially modular for the trafficking behavior of PINs and that the intracellular trafficking of PIN is plastic depending on cell type and developmental stage.

The fragile Fiber1 kinesin contributes to cortical microtubule-mediated trafficking of cell wall components.

The cell wall consists of cellulose microfibrils embedded within a matrix of hemicellulose and pectin. Cellulose microfibrils are synthesized at the plasma membrane, whereas matrix polysaccharides are synthesized in the Golgi apparatus and secreted. The trafficking of vesicles containing cell wall components is thought to depend on actin-myosin. Here, we implicate microtubules in this process through studies of the kinesin-4 family member, Fragile Fiber1 (FRA1). In an fra1-5 knockout mutant, the expansion rate of the inflorescence stem is halved compared with the wild type along with the thickness of both primary and secondary cell walls. Nevertheless, cell walls in fra1-5 have an essentially unaltered composition and ultrastructure. A functional triple green fluorescent protein-tagged FRA1 fusion protein moves processively along cortical microtubules, and its abundance and motile density correlate with growth rate. Motility of FRA1 and cellulose synthase complexes is independent, indicating that FRA1 is not directly involved in cellulose biosynthesis; however, the secretion rate of fucose-alkyne-labeled pectin is greatly decreased in fra1-5, and the mutant has Golgi bodies with fewer cisternae and enlarged vesicles. Based on our results, we propose that FRA1 contributes to cell wall production by transporting Golgi-derived vesicles along cortical microtubules for secretion.

A single pair of pharyngeal neurons functions as a commander to reject high salt in Drosophila melanogaster.

Salt is an essential nutrient for survival, while excessive NaCl can be detrimental. In the fruit fly, Drosophila melanogaster, internal taste organs in the pharynx are critical gatekeepers impacting the decision to accept or reject a food. Currently, our understanding of the mechanism through which pharyngeal gustatory receptor neurons (GRNs) sense high salt are rudimentary. Here, we found that a member of the ionotropic receptor family, Ir60b, is expressed exclusively in a pair of GRNs activated by high salt. Using a two-way choice assay (DrosoX) to measure ingestion volume, we demonstrate that IR60b and two coreceptors IR25a and IR76b, are required to prevent high salt consumption. Mutants lacking external taste organs but retaining the internal taste organs in the pharynx exhibit much higher salt avoidance than flies with all taste organs but missing the three IRs. Our findings highlight the vital role for IRs in a pharyngeal GRN to control ingestion of high salt.

A single pair of pharyngeal neurons functions as a commander to reject high salt in Drosophila melanogaster.

Salt (NaCl), is an essential nutrient for survival, while excessive salt can be detrimental. In the fruit fly, Drosophila melanogaster, internal taste organs in the pharynx are critical gatekeepers impacting the decision to accept or reject a food. Currently, our understanding of the mechanism through which pharyngeal gustatory receptor neurons (GRNs) sense high salt are rudimentary. Here, we found that a member of the ionotropic receptor family, Ir60b, is expressed exclusively in a pair of GRNs activated by high salt. Using a two-way choice assay (DrosoX) to measure ingestion volume, we demonstrate that IR60b and two co-receptors IR25a and IR76b are required to prevent high salt consumption. Mutants lacking external taste organs but retaining the internal taste organs in the pharynx exhibit much higher salt avoidance than flies with all taste organs but missing the three IRs. Our findings highlight the vital role for IRs in a pharyngeal GRN to control ingestion of high salt.

A dose-dependent bimodal switch by homologous Aux/IAA transcriptional repressors.

Combinatorial interactions between different regulators diversify and enrich the chance of transcriptional regulation in eukaryotic cells. However, a dose-dependent functional switch of homologous transcriptional repressors has rarely been reported. Here, we show that SHY2, an auxin/indole-3-acetic acid (Aux/IAA) repressor, exhibits a dose-dependent bimodal role in auxin-sensitive root-hair growth and gene transcription in Arabidopsis, whereas other Aux/IAA homologs consistently repress the auxin responses. The co-repressor (TOPLESS [TPL])-binding affinity of a bimodal Aux/IAA was lower than that of a consistently repressing Aux/IAA. The switch of a single amino acid residue in the TPL-binding motif between the bimodal form and the consistently repressing form switched their TPL-binding affinity and transcriptional and biological roles in auxin responses. Based on these data, we propose a model whereby competition between homologous repressors with different co-repressor-binding affinities could generate a bimodal output at the transcriptional and developmental levels.

Gut tumors in flies alter the taste valence of an anti-tumorigenic bitter compound.

The sense of taste is essential for survival, as it allows animals to distinguish between foods that are nutritious from those that are toxic. However, innate responses to different tastants can be modulated or even reversed under pathological conditions. Here, we examined whether and how the internal status of an animal impacts taste valence by using Drosophila models of hyperproliferation in the gut. In all three models where we expressed proliferation-inducing transgenes in intestinal stem cells (ISCs), hyperproliferation of ISCs caused a tumor-like phenotype in the gut. While tumor-bearing flies had no deficiency in overall food intake, strikingly, they exhibited an increased gustatory preference for aristolochic acid (ARI), which is a bitter and normally aversive plant-derived chemical. ARI had anti-tumor effects in all three of our gut hyperproliferation models. For other aversive chemicals we tested that are bitter but do not have anti-tumor effects, gut tumors did not affect avoidance behaviors. We demonstrated that bitter-sensing gustatory receptor neurons (GRNs) in tumor-bearing flies respond normally to ARI. Therefore, the internal pathology of gut hyperproliferation affects neural circuits that determine taste valence postsynaptic to GRNs rather than altering taste identity by GRNs. Overall, our data suggest that increased consumption of ARI may represent an attempt at self-medication. Finally, although ARI's potential use as a chemotherapeutic agent is limited by its known toxicity in the liver and kidney, our findings suggest that tumor-bearing flies might be a useful animal model to screen for novel anti-tumor drugs.

Functional identification of the phosphorylation sites of Arabidopsis PIN-FORMED3 for its subcellular localization and biological role.

Directional cell-to-cell movement of auxin is mediated by asymmetrically localized PIN-FORMED (PIN) auxin efflux transporters. The polar localization of PINs has been reported to be modulated by phosphorylation. In this study, the function of the phosphorylation sites of the PIN3 central hydrophilic loop (HL) was characterized. The phosphorylation sites were located in two conserved neighboring motifs, RKSNASRRSF(/L) and TPRPSNL, where the former played a more decisive role than the latter. Mutations of these phosphorylatable residues disrupted in planta phosphorylation of PIN3 and its subcellular trafficking, and caused defects in PIN3-mediated biological processes such as auxin efflux activity, auxin maxima formation, root growth, and root gravitropism. Because the defective intracellular trafficking behaviors of phospho-mutated PIN3 varied according to cell type, phosphorylation codes in PIN3-HL are likely to operate in a cell-type-specific manner.

The M3 phosphorylation motif has been functionally conserved for intracellular trafficking of long-looped PIN-FORMEDs in the Arabidopsis root hair cell.

BACKGROUND: PIN-FORMED (PIN) efflux carriers contribute to polar auxin transport and plant development by exhibiting dynamic and diverse asymmetrical localization patterns in the plasma membrane (PM). Phosphorylation of the central hydrophilic loop (HL) of PINs has been implicated in the regulation of PIN trafficking. Recently, we reported that a phosphorylatable motif (M3) in the PIN3-HL is necessary for the polarity, intracellular trafficking, and biological functions of PIN3. In this study, using the root hair system for PIN activity assay, we investigated whether this motif has been functionally conserved among long-HL PINs. RESULTS: Root hair-specific overexpression of wild-type PIN1, 2, or 7 greatly inhibited root hair growth by depleting auxin levels in the root hair cell, whereas overexpression of M3 phosphorylation-defective PIN mutants failed to inhibit root hair growth. Consistent with this root hair phenotype, the PM localization of M3 phosphorylation-defective PIN1 and PIN7 was partially disrupted, resulting in less auxin efflux and restoration of root hair growth. Partial formation of brefeldin A-compartments in these phosphorylation-mutant PIN lines also suggested that their PM targeting was partially disrupted. On the other hand, compared with the PIN1 and PIN7 mutant proteins, M3-phosphorylation-defective PIN2 proteins were almost undetectable. However, the mutant PIN2 protein levels were restored by wortmannin treatment almost to the wild-type PIN2 level, indicating that the M3 motif of PIN2, unlike that of other PINs, is implicated in PIN2 trafficking to the vacuolar lytic pathway. CONCLUSIONS: These results suggest that the M3 phosphorylation motif has been functionally conserved to modulate the intracellular trafficking of long-HL PINs, but its specific function in trafficking has diverged among PIN members.

Phospholipase A(2) is required for PIN-FORMED protein trafficking to the plasma membrane in the Arabidopsis root.

Phospholipase A(2) (PLA(2)), which hydrolyzes a fatty acyl chain of membrane phospholipids, has been implicated in several biological processes in plants. However, its role in intracellular trafficking in plants has yet to be studied. Here, using pharmacological and genetic approaches, the root hair bioassay system, and PIN-FORMED (PIN) auxin efflux transporters as molecular markers, we demonstrate that plant PLA(2)s are required for PIN protein trafficking to the plasma membrane (PM) in the Arabidopsis thaliana root. PLA(2)alpha, a PLA(2) isoform, colocalized with the Golgi marker. Impairments of PLA(2) function by PLA(2)alpha mutation, PLA(2)-RNA interference (RNAi), or PLA(2) inhibitor treatments significantly disrupted the PM localization of PINs, causing internal PIN compartments to form. Conversely, supplementation with lysophosphatidylethanolamine (the PLA(2) hydrolytic product) restored the PM localization of PINs in the pla(2)alpha mutant and the ONO-RS-082-treated seedling. Suppression of PLA(2) activity by the inhibitor promoted accumulation of trans-Golgi network vesicles. Root hair-specific PIN overexpression (PINox) lines grew very short root hairs, most likely due to reduced auxin levels in root hair cells, but PLA(2) inhibitor treatments, PLA(2)alpha mutation, or PLA(2)-RNAi restored the root hair growth of PINox lines by disrupting the PM localization of PINs, thus reducing auxin efflux. These results suggest that PLA(2), likely acting in Golgi-related compartments, modulates the trafficking of PIN proteins.

Chemosensory detection of aversive concentrations of ammonia and basic volatile amines in insects.

Basic volatiles like ammonia are found in insect environments, and at high concentrations cause an atypical action potential burst, followed by inhibition in multiple classes of olfactory receptor neurons (ORNs) in Drosophila melanogaster. During the period of inhibition, ORNs are unable to fire action potentials to their ligands but continue to display receptor potentials. An increase in calcium is also observed in antennal cells of Drosophila and Aedes aegypti. In the gustatory system, ammonia inhibits sugar and salt responses in a dose-dependent manner. Other amines show similar effects in both gustatory and olfactory neurons, correlated with basicity. The concentrations that inhibit neurons reduce proboscis extension to sucrose in Drosophila. In Aedes, a brief exposure to volatile ammonia abolishes attraction to human skin odor for several minutes. These findings reveal an effect that prevents detection of attractive ligands in the olfactory and gustatory systems and has potential in insect control.

Differential auxin-transporting activities of PIN-FORMED proteins in Arabidopsis root hair cells.

The Arabidopsis (Arabidopsis thaliana) genome includes eight PIN-FORMED (PIN) members that are molecularly diverged. To comparatively examine their differences in auxin-transporting activity and subcellular behaviors, we expressed seven PIN proteins specifically in Arabidopsis root hairs and analyzed their activities in terms of the degree of PIN-mediated root hair inhibition or enhancement and determined their subcellular localization. Expression of six PINs (PIN1-PIN4, PIN7, and PIN8) in root hair cells greatly inhibited root hair growth, most likely by lowering auxin levels in the root hair cell by their auxin efflux activities. The auxin efflux activity of PIN8, which had not been previously demonstrated, was further confirmed using a tobacco (Nicotiana tabacum) cell assay system. In accordance with these results, those PINs were localized in the plasma membrane, where they likely export auxin to the apoplast and formed internal compartments in response to brefeldin A. These six PINs conferred different degrees of root hair inhibition and sensitivities to auxin or auxin transport inhibitors. Conversely, PIN5 mostly localized to internal compartments, and its expression in root hair cells rather slightly stimulated hair growth, implying that PIN5 enhanced internal auxin availability. These results suggest that different PINs behave differentially in catalyzing auxin transport depending upon their molecular activity and subcellular localization in the root hair cell.