PIN Proteins Perform a Rate-Limiting Function in Cellular Auxin Efflux

Auxin, a regulatory compound, plays a major role in the spatial and temporal coordination of plant development (1–3). The directional active cell-to-cell transport controls asymmetric auxin distribution, which underlies multiple patterning and differential growth processes (4–7). Genetic approaches in Arabidopsis thaliana identified candidate genes coding for regulators of auxin transport, among them permease-like AUX1 (8), plant-specific PIN proteins (9) (fig. S1), and homologs of human multiple drug resistance transporters PGP1 and PGP19 (10, 11). PGP1 has been shown to mediate the efflux of auxin from Arabidopsis protoplasts and heterologous systems such as yeast and HeLa cells (12). Similarly, PIN2 in yeast conferred decreased retention of structural auxin analogs (13, 14). Plants defective in PIN function show altered auxin distribution and diverse developmental defects, all of which can be phenocopied by chemical inhibition of auxin efflux (1, 4–7, 9). All results demonstrate that PINs are essential components of the auxin transport machinery, but the exact mechanism of their action remains unclear.

Studies of the molecular function of PINs have been hampered mainly by the technical inability to quantitatively assess auxin flow across the plasma membrane (PM) in a multicellular system. We therefore established Arabidopsis cell suspension culture from the XVE-PIN1 line, in which we placed the PIN1 sequence under control of the estradiol-inducible promoter (15). Treatment with estradiol led to the activation of PIN1 expression as shown by the coexpressed green fluorescent protein (GFP) reporter and reverse transcription polymerase chain reaction (RT-PCR) of PIN1 in seedlings (Fig. 1A) and cultured cells (fig. S2). In estradiol-treated XVE-PIN1 cells, the overexpressed PIN1 was localized at the PM (Fig. 1, B and C). The synthetic auxin naphthalene-1-acetic acid (NAA) enters cells easily by diffusion and is a poor substrate for active uptake but an excellent substrate for active efflux (16). Therefore, change in accumulation of radioactively labeled NAA inside cells provides a measure of the rate of auxin efflux from cells. Untreated XVE-PIN1 cells as well as nontransformed cells displayed [³H]NAA accumulation kinetics indicative of saturable auxin efflux and sensitive to a well-established (1, 9) noncompetitive inhibitor of auxin efflux: 1-naphthylphthalamic acid (NPA) (Fig. 1D). Estradiol did not influence control cells but led to substantial decrease of [³H]NAA accumulation in XVE-PIN1 cells (Fig. 1, D and E). This demonstrates that PIN1 overexpression leads to the stimulation of efflux of auxin from Arabidopsis cultured cells.

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Arabidopsis cultured cells are not sufficiently friable to be useful in transport assays. Instead, we used tobacco BY-2 cells, a well-established model for quantitative studies of cellular auxin transport (17). PIN7, the most representative member of the subfamily including PIN1, PIN2, PIN3, PIN4, PIN6, and PIN7 (fig. S1), was placed under the control of a dexamethasone (DEX)–inducible system (18) and stably transformed into BY-2 cells. The resulting line (GVG-PIN7) showed up-regulation of PIN7 expression as early as 2 hours after DEX treatment and the up-regulated PIN7 protein was detected at the PM (Fig. 2A). Nontransformed cells displayed saturable, NPA-sensitive [³H]NAA efflux, which was unaffected by DEX (Fig. 2B). Induction of expression of PIN7 or its close (PIN4) and the most distant (PIN6) homologs (fig. S1) resulted in a decrease in [³H]NAA accumulation, to roughly half of the original level (Fig. 2C). The kinetics of NAA efflux after the initial loading of BY-2 cells (Fig. 2D), as well as displacement curves using competitive inhibition by nonlabeled NAA (fig. S3A), clearly confirm that PIN7 overexpression stimulates saturable efflux of auxin from cells. The efflux of other auxins—such as synthetic 2,4-dichlorophenoxyacetic acid (2,4-D) or naturally occurring indole-3-acetic acid (IAA), but not its precursor tryptophan—was also stimulated (Fig. 2, E and G). The PIN7-dependent efflux of all auxins was NPA sensitive (Fig. 2G), competitively inhibited by nonlabeled NAA, and unaffected by the structurally related but biologically inactive weak organic acid, benzoic acid (BeA) (fig. S3B). Furthermore, the increasing levels of induced PIN7, as achieved with the use of different concentrations of DEX for induction, and monitored by dot blot, clearly correlated with the gradual increase in [³H]NAA efflux (Fig. 2F). These data imply that different PIN proteins are rate-limiting factors in NPA-sensitive, saturable efflux of auxins from BY-2 cells. This similarity in the molecular function of PINs, together with the diversity in their regulation, provides a basis for their complex functional redundancy observed in planta (6, 19, 20).

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The evidence from cultured cells shows that PIN proteins are key rate-limiting factors in cellular auxin efflux. This approach, however, cannot distinguish whether PINs play a catalytic role in auxin efflux or act as positive regulators of endogenous plant auxin efflux catalysts. To address this issue, we used a nonplant system: Human HeLa cells contain neither PIN-related genes nor auxin-related machinery and allow efficient heterologous expression of functional eukaryotic PM proteins (21). We transfected HeLa cells with PIN7 and its more distant homolog PIN2. Transfected cells showed strong PIN expression (Fig. 3A), which resulted in a substantial stimulation of net efflux of natural auxin [³H]IAA, compared with empty vector controls (Student's t test: P < 0.001) (Fig. 3B). Efflux of [³H]BeA was also stimulated but to a lesser extent. These data show that PIN proteins are capable of stimulating cellular auxin efflux in the heterologous HeLa cell system, albeit with decreased substrate specificity.

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To test the role of PIN proteins in another evolutionarily distant nonplant system, we used yeast (Saccharomyces cerevisiae). PIN2 and PIN7 were expressed in yeast and showed localization at the PM (Fig. 3A). Kinetics of relative [³H]IAA retention demonstrated that expression of the PINs led to a substantial increase in IAA efflux (Fig. 3C). Efflux assays in conjunction with control experiments, including testing metabolically less active yeast in the stationary phase, or after glucose starvation (Fig. 3D), confirmed an active PIN-dependent export of IAA and, to a lesser extent, of BeA from yeast (Fig. 3C and fig. S4B). To test the requirements of the subcellular localization for PIN2 action in yeast, we performed a mutagenesis of the PIN2 sequence to isolate mistargeted mutants. One of the mutations, which changed serine-97 to glycine (pin2Gly97), led to the localization of pin2Gly97 in intracellular compartments (Fig. 3A). When tested in the [³H]IAA efflux assay (fig. S4A), pin2Gly97 failed to mediate auxin efflux but rather increased [³H]IAA accumulation inside cells (Fig. 3D). This shows that pin2Gly97 is still functional but fails to mediate auxin efflux, suggesting importance of PIN localization at PM. Overall, the results suggest that in yeast as well, PM-localized PIN proteins mediate, although with decreased specificity, a saturable efflux of auxin.

A role in auxin efflux has also been reported recently for PGP1 and, in particular, PGP19 proteins of Arabidopsis (12). PIN and PGP proteins seem to have a comparable effect on mediating auxin efflux in yeast and HeLa cells, but the genetic interference with their function in Arabidopsis has distinctive effects on development. All aspects of the pin mutant phenotypes can be mimicked by chemical interference with auxin transport (4–7, 9). In contrast, pgp1/pgp19 double mutants show strong but entirely different defects (10, 11), which cannot be phenocopied by auxin transport inhibitors.

To compare the roles of PINs and PGPs in auxin efflux, we constructed the GVG-PGP19:HA (hemagglutinin) cell line of BY-2. DEX treatment led to the up-regulation of PGP19:HA protein, which was detected at the PM (Fig. 4A), and to a decrease in [³H]NAA accumulation, similar to that observed in the GVG-PIN4, GVG-PIN6, and GVG-PIN7 lines (Fig. 4B, compare with Fig. 2C). BeA did not interfere with [³H]NAA accumulation and [³H]Trp accumulation did not change after DEX treatment. However, compared with PIN-mediated auxin efflux, the PGP19-mediated NAA efflux was notably less sensitive to NPA. Whereas PIN-mediated transport was completely inhibited by NPA, about 20% of PGP19-dependent transport was NPA insensitive (Fig. 4C).

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To address whether PIN action in planta requires PGP1 and PGP19 proteins, we analyzed effects of PIN1 overexpression on plant development in pgp1/pgp19 double mutants. PIN1 overexpression in XVE-PIN1 led to pronounced defects in root gravitropism, which could be detected within 4 hours after estradiol treatment. Quantitative evaluation of reorientation of root growth revealed that PIN1 overexpression in pgp1/pgp19 had the same effects (Fig. 4D). These data show that PIN1 action on plant development does not strictly require function of PGP1 and PGP19 proteins, and they suggest that PINs and PGPs molecularly characterize distinct auxin transport systems. This is also supported by evidence that PIN2 mediates auxin efflux in yeast, which is known to lack homologs to Arabidopsis PGP proteins (21). It is still unclear whether these two auxin transport machineries act in planta entirely independently or in a coordinated fashion.

Rate-limiting, saturable, and specific action of PIN proteins in mediating auxin movement across the PM out of plant cells largely clarifies a role of PIN proteins in intercellular auxin transport. Furthermore, the polar, subcellular PIN localization provides a vectorial component to the directional auxin flow (22). Therefore, transport function of PINs together with their asymmetric subcellular localization defines directional local auxin distribution underlying different developmental processes.