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

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Science  12 May 2006:
Vol. 312, Issue 5775, pp. 914-918
DOI: 10.1126/science.1123542

Abstract

Intercellular flow of the phytohormone auxin underpins multiple developmental processes in plants. Plant-specific pin-formed (PIN) proteins and several phosphoglycoprotein (PGP) transporters are crucial factors in auxin transport–related development, yet the molecular function of PINs remains unknown. Here, we show that PINs mediate auxin efflux from mammalian and yeast cells without needing additional plant-specific factors. Conditional gain-of-function alleles and quantitative measurements of auxin accumulation in Arabidopsis and tobacco cultured cells revealed that the action of PINs in auxin efflux is distinct from PGP, rate-limiting, specific to auxins, and sensitive to auxin transport inhibitors. This suggests a direct involvement of PINs in catalyzing cellular auxin efflux.

Auxin, a regulatory compound, plays a major role in the spatial and temporal coordination of plant development (13). The directional active cell-to-cell transport controls asymmetric auxin distribution, which underlies multiple patterning and differential growth processes (47). 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, 47, 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.

Fig. 1.

PIN1-dependent auxin efflux in Arabidopsis cultured cells. (A) Up-regulation of PIN1 expression in XVE-PIN1 Arabidopsis seedlings after estradiol (EST) treatment (1 μM, 4 hours). The expression of coupled GFP reporter (green) and RT-PCR of PIN1 [PGP19 expression was used as a control (cont.)] are shown. Scale bars, 3 mm. (B and C) Anti-PIN1 immunostaining (red) at the PM of XVE-PIN1 cultured cells after EST treatment (1 μM, 24 hours) (C). There was no signal in the untreated control (B). Nuclear counterstain is shown in blue. Scale bars, 10 μm. (D) Auxin accumulation in Arabidopsis wild-type cells. NPA (10 μM) increased [3H]NAA accumulation inside cells, demonstrating inhibition of auxin efflux. EST treatment (1 μM, 24 hours) had no effect on [3H]NAA accumulation. (E) [3H]NAA accumulation kinetics in XVE-PIN1 cells, demonstrating PIN1-dependent stimulation of NAA efflux after PIN1 overexpression. Error bars show SEM (n = 4); where error bars are not shown, the error was smaller than the symbols.

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).

Fig. 2.

PIN-dependent auxin efflux in BY-2 tobacco cultured cells. (A) Inducible PIN7 expression in GVG-PIN7 tobacco cells. PIN7 immunostaining (green) is shown at the PM after DEX treatment (24 hours; 1 μM) but not in the untreated control; RT-PCR of PIN7 was conducted within 24 hours of DEX treatment (1 μM). Nuclear counterstain is shown in blue. Scale bars, 40 μm. (B) Auxin accumulation in BY-2 control cells. NPA (10 μM) increased [3H]NAA accumulation inside cells, demonstrating inhibition of auxin efflux. DEX treatment (1 μM, 24 hours) had no effect on [3H]NAA accumulation. (C) [3H]NAA accumulation kinetics in GVG-PIN4, GVG-PIN6, and GVG-PIN7 cells demonstrating PIN4-, PIN6-, and PIN7-dependent stimulation of NAA efflux. Noninduced control is shown only for PIN7; those for PIN4 and PIN6 were within the range ±8% of the values for PIN7. Data are expressed as a percentage of noninduced control at 30 min after application of labeled [3H]NAA. (D) Induced GVG-PIN7 cells showed decreased retention of [3H]NAA compared with noninduced control. (E) Accumulation kinetics in induced GVG-PIN7 cells revealed PIN7-dependent stimulation of [3H]IAA and [3H]2,4-D efflux. (F) Treatments with increasing concentrations of DEX led to gradually higher levels of PIN7 in GVG-PIN7 cells, as determined by dot blot (top) and to concomitant decrease of [3H]NAA accumulation. (G) NPA inhibition of both endogenous and PIN7-dependent efflux of [3H]NAA, [3H]2,4-D, and [3H]IAA. PIN7 overexpression or NPA treatment did not affect accumulation of related compound, [3H]Trp. Open bars, noninduced cells; gray bars, induced cells. For all experiments, error bars show SEM (n = 4); where error bars are not shown, the error was smaller than the symbols.

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.

Fig. 3.

PIN-dependent auxin efflux in mammalian and yeast cells. (A) PIN2:HA and PIN7:HA expression in HeLa and yeast. Anti-HA immunostaining detected PIN2:HA and PIN7:HA at the PM of transfected but not control (empty vector) HeLa cells (top). Anti-PIN2 immunostaining detected PIN2 at the PM and pin2Gly97 in intracellular compartments, compared with empty vector controls (bottom). Scale bars, 2 μm. (B) Transfected HeLa cells display PIN2- and PIN7-dependent net efflux of [3H]IAA and to a smaller extent also of [3H]BeA. dpm, disintegration per minute. (C) The kinetics of [3H]IAA efflux. PIN2 stimulated saturable [3H]IAA efflux in yeast JK93da strain. (D) [3H]IAA retention measured 10 min after loading: PIN2 and PIN7 mediated [3H]IAA efflux; pin2Gly97 failed to mediate efflux but increased [3H]IAA retention. Yeast in stationary phase or without glucose showed much less [3H]IAA efflux. Error bars show SEM (n = 4).

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 (47, 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).

Fig. 4.

Requirement of PGP function for PIN role in auxin efflux. (A) Inducible PGP19 expression in GVG-PGP19:HA tobacco cells. PGP19:HA immunostaining (red) at PM after DEX treatment (24 hours, 1 μM) is shown; no PGP19:HA immunostaining was present in the untreated control. An anti-HA immunoblot was conducted after 24 hours of DEX (1 μM) treatment. Nuclear counterstain is shown in blue. Scale bars, 40 μm. (B) [3H]NAA accumulation decreased upon PGP19 expression, revealing function in auxin efflux in BY-2 cells. (C) Different sensitivities of PIN7- and PGP19-dependent [3H]NAA efflux to NPA treatment (10 μM, 20 min) in GVG-PIN7 and GVG-PGP19:HA cells (23). (D) Root gravitropism in XVE-PIN1 seedlings. PIN1 overexpression (4 hours, 4 μM EST) led to gravitropic defects in pgp1/pgp19 mutants in contrast to gravitropic growth of EST-treated nontransformed wild-type (WT) and pgp1/pgp19 seedlings. Root gravitropism was scored 12 hours after gravity stimulation (n > 40). Scale bar, 3 mm. For (B) and (C), error bars show SEM (n = 4); where error bars are not shown, the error was smaller than the symbols.

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.

Supporting Online Material

www.sciencemag.org/cgi/content/full/1123542/DC1

Materials and Methods

Figs. S1 to S4

References

References and Notes

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