Relocalization of the PIN 1 Auxin Efflux Facilitator Plays a Role in Phototropic Responses 1 [ w ]

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). Molecular genetic and physiological evidence indicates that PIN1 is required for transport of auxin from shoot to root apices (Okada et al., 1991; Friml and Palme, 2002), whereas immunohistochemical studies localize PIN1 to the lower ends of xylemassociated cells in both shoots and roots (Gälweiler et al., 1998). More recently, asymmetric PIN1 localization in root tips has been shown to involve dynamic cycling mediated by the ARF-GEF GNOM (Geldner et al., 2001, 2003). Surprisingly, although disruption of MDR/PGP genes results in decreased polar auxin transport (Noh et al., 2001; Geisler et al., 2003; Multani et al., 2003) and decreased free auxin content in lower shoot and root tissues (J.J. Blakeslee and A.S. Murphy, unpublished data), mdr1 pgp1 mutant hypocotyls exhibit exaggerated tropic bending (Noh et al., 2003). According to the Cholodny-Went hypothesis (for summary, see Went, 1974), tropic bending is mediated by lateral redistribution of auxin near the site of tropic stimulus. Our report suggested that the enhanced tropic bending observed in mdr/pgp mutants resulted from decreased vertical auxin transport and a consequent increase of lateral auxin bias. However, it was not clear whether the perturbation of PIN1 localization in mdr/pgp mutants was due to disruption of an immediate interaction required for asymmetric localization of the PIN1 protein or from cumulative developmental defects resulting from altered auxin transport. The extent to which altered localized auxin levels might contribute to PIN1 delocalization in tissues where AtMDR1 and AtPGP1 are not expressed was also not determined. To determine whether PIN1 delocalization similar to that seen in hyper-phototropic mdr/pgp mutants also plays a role in the normal phototropic response, we investigated changes in PIN1 localization after initiation of the first positive phototropic curvature in wild-type (WT) seedlings responding to 450 nm of blue light. When illuminated with directional blue light, Arabidopsis seedlings exhibit a phototropic bending response that involves two steps—an initial halt in vertical growth and subsequent initiation of bending (Parks et al., 2001). A signal transduction pathway initiated by phototropin perception of blue light (Briggs et al., 2001) and modulated by both Ca (Harada et al., 2003; Stoelzle et al., 2003) and protein signaling components (Motchoulski and Liscum, 1999) has been shown to mediate the bending response. Although directional blue light is thought to be perceived in the upper portion of the hypocotyl (Parks et al., 2001), bending manifests in the midhypocotyl region and is thought to be regulated by auxin efflux through the laterally oriented PIN3 auxin efflux facilitator (Friml et al., 2002). When WT seedlings were grown in the dark, where basal localization of PIN1 was observed (Fig. 1A), and subsequently exposed to unidirectional blue light (0.5 mol m 2 s , 450 nm, for 1.5 h), we observed phototropic bending of seedlings similar to that previously described (Briggs et al., 2001; Friml et al., 2002). After blue light stimulus, tissues were rapidly fixed, and both PIN1 and PIN3 proteins were localized by immunofluorescence microscopy. Although no changes in PIN3 localization could be detected (data not shown), we observed PIN1 delocalization (Fig. 1B) in the mid-hypocotyl region where phototropic bending occurs and has been shown previously to accumulate auxin as part of that response (Friml et al., 2002). A gradient in PIN1 delocalization was observed, with a disruption of basal localization of PIN1 on the side of the seedling distal to the light source (Fig. 1B). To confirm that this was a blue light-dependent phenomenon, we subjected phot1 mutants to the same blue light treatment. Although it was more difficult to visualize PIN1 by immunofluorescence in Col-0 hypocotyls, patterns of PIN1 localization in dark-grown (Fig. 1C) and blue light-treated Col-0 hypocotyls resembled those observed in Ws (Fig. 1A). However, PIN1 localization in blue light-treated phot1 mutant hypocotyls (Fig. 1D) resembled that seen in untreated WT Col-0 hypocotyls (Fig. 1C), demonstrating the strict blue light dependence of the observed changes in 1 This work was supported by the National Science Foundation (grant no. 0132803). * Corresponding author; e-mail murphy@purdue.edu; fax 765– 494 – 0391. [w] The online version of this article contains Web-only data. http://www.plantphysiol. org/cgi/doi/10.1104/pp.103. 031690.