Ranocha P , Dima O , Nagy R , Felten J , Corratgé-Faillie C , Novák O , Morreel K , Lacombe B , Martinez Y , Pfrunder S , Jin X , Renou JP , Thibaud JB , Ljung K , Fischer U , Martinoia E , Boerjan W , Goffner D .

	Figure 1. WAT1 coexpression vicinity network.The most highly co-regulated genes with WAT1 as identified with the Arabidopsis Coexpression Data Mining Tools from the University of Leeds21 (http://www.Arabidopsis.leeds.ac.uk/act/coexpanalyser.php). Nodes represent genes; edge width indicates whether two given genes are co-expressed above a certain mutual rank threshold56. Nodes are colour coded to reflect the level of induction by auxin of the corresponding genes according to the Genevestigator22 (https://www.genevestigator.com) and the Bio-Array Resource57 (http://www.bar.utoronto.ca) websites.
	Figure 2. WAT1 is induced by auxin.(a) Reverse transcriptase (RT)–PCR analysis of WAT1 expression in 10-day-old wild-type plantlets transferred for 2 h on MS medium with or without 1 μM IAA before RNA extraction. The amount of cDNA template in each RT–PCR reaction was normalized to the signal from the actin-encoding ACT2 gene, and primers were designed to rule out the amplification of genomic DNA. Histograms represent WAT1 expression levels normalized versus ACT2 expression levels. Mean±s.d., n=3 biological replicates. (b) GUS activity in the aerial portion (top) and roots (bottom) of 10-day-old ProWAT1:GUS plantlets transferred for 2 h on MS medium with or without 1 μM IAA before staining.
	Figure 3. Local auxin application rescues deficiency in secondary cell wall formation in wat1 mutants.Phloroglucinol-HCl staining of transverse stem sections, lignified cell walls stained red. Lanolin±auxin was applied to the second oldest internode of the inflorescence stem. Stems were sectioned 10 days after application. Sections are representative of data from two experimental replicates each with six individuals per genotype and treatment. Scale bars, 50 μm.
	Figure 4. WAT1 is a vacuolar auxin transporter.(a) Co-localization of WAT1-GFP with known fluorescent membrane markers. F1 generation of crosses between p35S::WAT1-GFP and plants expressing either YFP-VAM711, a tonoplast marker (top row), YFP-RabG3f, a late endosome marker (central row) or YFP-NPSN12, a plasma membrane marker (bottom row). Co-localization in root epidermis cells in the elongation zone of 5-day-old Arabidopsis seedlings is shown. Note strong co-localization of WAT1-GFP with the tonoplast marker, partial co-localization with the late endosome marker and weak co-localization with the plasma membrane marker. Weak signal of WAT1-GFP at the plasma membrane (arrows). Scale bars, 10 μm. (b), Time-dependent transport of auxin in vacuoles isolated from wild-type plants and wat1 mutants in the absence of NH4Cl. The values represent means of five replicates, and the error bars stand for ±s.e.m. Simplified transport experiments with five replicates each at short and long time points were performed four times; their outcome is in line with the extended time course experiment. (c) Same as b, except in the presence of NH4Cl and the omission of Mg-ATP.
	Figure 5. Auxin fractionation after vacuole uptake.Auxin is not converted within vacuoles. Isolated vacuoles were incubated in the presence of 14C-auxin (IAA) for 20 min and subsequently removed from the incubation medium as described for the transport studies. The water phase containing the vacuoles was subjected to a HPLC run, fractions were collected and the radioactivity present in each fraction was detected. A comparison of panel a, where non-radioactive auxin was detected at 29 min, with panel b representing the radioactivity detected in each fraction, shows that the radioactivity appears at a similar retention time as the authentic auxin. The slight shift is because of the collection of the fractions that occurred after the ultraviolet detector. WVL, wavelength. For more details see Methods.
	Figure 6. WAT1 confers auxin efflux to yeast cells and Xenopus oocytes.(a) WAT1 transcript levels in yeast transformed either with the empty pDR196 vector or with pDR196-WAT1. Actin (ACT1) primers were used as control. (b) Subcellular localization in yeast of GFP-tagged WAT1. Scale bar, 1 μm. (c) 3H-IAA transport assay in yeast. Closed circles, yeasts expressing WAT1; open squares, control yeasts containing the empty vector. Mean±s.d., n=3 biological replicates. (d) Net accumulation in 15 min of 3H-IAA in WAT1-expressing yeast (relatively to control yeast containing the empty vector) in the presence of 500 μM potential non-radiolabelled competitors. Mean±s.d., n=3 biological replicates. (e) 3H-tryptophan transport assay in yeast. Same as c. (f) Expression of WAT1 increases auxin uptake in Xenopus oocytes. Auxin uptake was measured by the incorporation of 3H-labelled IAA (see Methods). Data are displayed as mean values (±s.d.) of auxin influx in oocytes injected either with deionized water (‘control’, n=10) or with WAT1 cRNAs (‘WAT1’, n=10). *, a Student’s test (threshold 0.98) showed that ‘WAT1’ mean value is statistically different from the ‘control’.

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