Supporting Online Material for

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1 Supporting Online Material for Polar PIN Localization Directs Auxin Flow in Plants Justyna Wiśniewska, Jian Xu, Daniela Seifertová, Philip B. Brewer, Kamil Růžička, Ikram Blilou, David Roquie, Eva Benková, Ben Scheres, Jiří Friml* *To whom correspondence should be addressed. This PDF file includes: Materials and Methods Fig. S1 References and Notes Published 6 April 2006 on Science Express DOI: /science

2 Wi niewska et al. Supporting online materials Abstract Polar flow of the phytohormone auxin requires plasma membrane-associated PIN proteins and underlies multiple developmental processes. Here we address the importance of the polarity of subcellular PIN localization for the directionality of auxin transport. Expression of different PINs in the root epidermis revealed the importance of PIN polar positions for directional auxin flow and root gravitropic growth. Interfering with sequence-embedded polarity signals directly demonstrates that PIN polarity is a primary factor determining the direction of auxin flow in meristematic tissues. This provides a crucial piece in the puzzle of how auxin flow can be redirected via rapid changes in PIN polarity. Material and Methods Material and growth conditions. Arabidopsis seedlings were grown in a 16 hours light/8 hours dark cycle at C on 0.5 x MS with sucrose as described (1). The following mutants, transgenic plants and constructs have been described previously: eir1-1, agr1, PIN2::PIN2:HA, DR5rev::GFP (1-5). The PIN2::PIN1,3,4:HA constructs were generated by fusion of the PIN2 promoter (1302 bp) and the PIN1 (At1g73590), PIN3 (At1g70940) and PIN4 (At2g01420) cdnas with the 9-amino-acid HA epitope tag at the C-terminus analogically to PIN2::PIN2:HA (5), and introduced into wild type and pin2 (eir1-1, agr1) mutants. The PIN1::PIN1:GFP-3 was generated by insertion of EGFP with linkers (AGAGAGAG-EGFP-AAAAAAAAAA) at position 1437 of the PIN1 genomic fragment (nucleotides to 4278 from ATG). PIN1::PIN1:GFP-2 was generated by

3 insertion of EGFP with linkers into PIN1 genomic fragment at position 651. The resulting fusions were cloned into the pgreenii-0229 vector ( and the functionality of both constructs was verified by the complementation of the pin1 mutant phenotype. PIN2::PIN1:GFP-2 and PIN2::PIN1:GFP-3 were generated from PIN1::PIN1:GFP-2 and PIN1::PIN1:GFP-3 by cloning the PIN1:GFP fusions (from ATG to the stop codon) into pgreenii-0229 between a PIN2 promoter (2171bp) and NOS terminator. The resulting constructs were transformed into wild type and pin2 (eir1-1) mutants. Localization analysis. Immunolocalizations in Arabidopsis were performed as described (6). For HA- and GFP-tagged PIN proteins, colocalization of anti-pin and anti-ha or anti-gfp were always performed to confirm the identity of the proteins. The following antibodies and dilutions were used: anti- PIN1 (7) (1: 500), -PIN2 (8) (1: 500), -PIN3 (9) (1: 50) and -PIN4 (10) (1:400), -HA (mouse) (Babco, 1:1000), -GFP (mouse) (Roche, 1:1000); and FITC- and CY3-conjugated anti-rabbit (1:200) or anti-mouse (1:500) secondary antibodies (Dianova). Phenotype analysis and microscopy. Arabidopsis root gravitropism assay (after 24 hours gravity stimulation in dark) (8) and gravitropic index (GI) evaluation (11) were performed in 5 day old seedlings. Root curvature and root lengths were measured using winprogs image J (11). Always, at least 30 roots of two independent lines in pin2 (eir1-1 and agr1) mutant backgrounds were analyzed. Results are presented as means with standard deviations and all claimed comparisons are based on t-test statistical evaluations. For confocal laser scanning microscopy, a Leica TCS SP2 (Arabidopsis) or Leica SP AOBS (BY-2) were used. Images were processed in Adobe Photoshop. Supporting References and Notes

4 1. E. Benková et al., Cell 115, 591 (2003). 2. J. Friml et al., Nature 426, 147 (2003). 3. R. Chen et al., Proc. Natl. Acad. Sci. USA 95, (1998). 4. C. Luschnig, R. A. Gaxiola, P. Grisafi, G.R Fink, Genes Dev. 12, 2175 (1998). 5. A. Vieten et al., Development 132, 4521 (2005). 6. J. Friml et al., Plant J. 34, 115 (2003). 7. T. Paciorek et al., Nature 435, 1251 (2005). 8. Generously provided by Christian Luschnig. 9. J. Friml et al., Nature 415, 806 (2002). 10. J. Friml et al., Cell 108, 661 (2002). 11. A. Grabov et al., New Phytol. 165, 641 (2005). Supporting Figure Legends Fig. S1. (A-G) PIN2:HA (A), PIN1:HA (B), PIN1:GFP-3 (C), PIN3:HA (D), PIN4:HA (E), PIN1:GFP-2 (F) and PIN2(red)/PIN1:GFP-2(green) (G) under PIN2 transcriptional control are expressed in epidermis and cortex of pin2 (eir1-1) mutants. In young cortex cells, all PIN proteins were predominantly localized to the lower side. In epidermal cells, PIN2, PIN2:HA and PIN1:GFP-3 are localized to the upper side, PIN3:HA and PIN4:HA to both sides and both PIN1:HA and PIN1:GFP-2 to the lower cell side. Anti-PIN (green: A, B, D, E; red: C, F, G); anti-ha (red: A, B, D, E) and anti-gfp (green: C, F, G)

5 colocalizations. (H) PIN1:GFP-3 under PIN1 transcriptional control shows basal localization in stele cells of pin1 mutant similar to endogenous PIN1 localization patterns. Arrowheads indicate polarity of PIN localization. Scale bars 5 µm.

6 A D epid. cor. epid. cor. B PIN2::PIN2:HA E PIN2::PIN3:HA C PIN2::PIN1:HA F PIN2::PIN4:HA G H PIN2::PIN1:GFP-3 PIN2::PIN1:GFP-2 PIN2 (wt) & PIN1:GFP-2 PIN1::PIN1:GFP-3