Volume 1, Issue 1, Pages 129-144 (January 2008) Disruptions in AUX1-Dependent Auxin Influx Alter Hypocotyl Phototropism in Arabidopsis  Stone Bethany B. , Stowe-Evans Emily L. , Harper Reneé M. , Celaya R. Brandon , Ljung Karin , Sandberg Göran , Liscum Emmanuel   Molecular Plant  Volume 1, Issue 1, Pages 129-144 (January 2008) DOI: 10.1093/mp/ssm013 Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 1 Hypocotyl Phototropic Responses of Wild-Type and Mutant Seedlings Exposed to Unidirectional UV-A Light and Unidirectional Blue Light, With and Without Red Light Co-Irradiation or Ethylene Pretreatment. (A) Phototropic curvatures of etiolated wild-type Col-O (WT), phot1 and nph4 seedlings exposed to 4 h of unidirectional BL (0.1 mmol m−2 s−1), unidirectional UV-A light (0.2 mmol m−2 s−1), or unidirectional BL supplemented with RL (1.6 mmol m−2 s−1) from above. Data represent mean response from a minimum of 60 seedlings from at least two independent replicate experiments. Error bars represent SE. (B) Phototropic curvatures of etiolated wild-type Col-O (WT), nph4 nph4map and nph4arf19 seedlings exposed to 4 h of unidirectional BL, unidirectional BL supplemented with RL, or unidirectional BL after growth on ACC (1 μM). Data represent mean response from a minimum of 80 seedlings from at least two independent replicate experiments. Error bars represent SE. Molecular Plant 2008 1, 129-144DOI: (10.1093/mp/ssm013) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 2 Identification of AUX1 as a Genetic Modifier of arf7 Phenotypes. (A) Root gravitropic phenotypes of wild-type Col-O (WT), aux1-7 and nph4map1/aux1-201 seedlings. Seedlings were grown in plates along the surface of the agar medium for 5 d in white light, then the plates were rotated 90° to induce a gravitropic response (downward curvature of the root; upward curvature of the hypocotyl). (B) Position of the aux1-201 Ala to Val missense mutation within a conserved motif of AUX/LAX family of auxin influx carriers. (C) Positions and identities of various aux1 missense alleles used in this study. Topology of AUX1 is adapted from Swarup et al. (2004) and shows the 11 predicted transmembrane (Tm) spans in linear sequence with Tm1 shown on the far left and Tm11 on the far right. Stars indicate the position of various missense alleles described in the box to the right. (D) Helical wheel projection of the second transmembrane span (Tm2) of AUX1. Hydrophobic residues are in black, hydrophilic residues are in red, and the Ala that is changed to Val in the aux1-201 allele is shown in grey. Projection adapted from Kerr and Bennett, 2007. Molecular Plant 2008 1, 129-144DOI: (10.1093/mp/ssm013) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 3 Hypocotyl Phototropism in Wild-Type and aux1 Mutant Seedlings. (A) Phototropic curvatures of wild-type Col-O (WT) and various aux1 missense mutants exposed to 4 h of unidirectional BL (0.1 mmol m−2 s−1). Although not all aux1 alleles are carried in the Col-O background (see Methods), only the Col-O response is shown, as other wild types responded similarly (data not shown). Data represent mean response from a minimum of 107 seedlings from at least two independent replicate experiments. Error bars represent SE. Asterisks denote responses significantly different from that observed in wild-type by Student's t-test (*, 0.01 > P > 0.001; **, P < 0.001). (B) Phototropic curvatures of nph4-null single and nph4aux1 double mutants exposed to 4 h of unidirectional BL supplemented with RL (1.6 mmol m−2 s−1) from above. Data represent mean response from a minimum of 75 seedlings from at least two independent replicate experiments. Error bars represent SE. Asterisks denote responses significantly different from that observed in the nph4-null single mutant by Student's t-test (*, 0.01 > P > 0.001; **, P < 0.001). Molecular Plant 2008 1, 129-144DOI: (10.1093/mp/ssm013) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 4 Influence of the Auxin Influx Inhibitor, 1-NOA, on Hypocotyl Phototropism in Wild-Type, nph4 and nph4aux1-201 Seedlings. Seedlings were grown for 3 d in darkness on agar-solidified medium containing the indicated concentration of 1-NOA, then exposed to 4 h of unidirectional BL (0.1 mmol m−2 s−1). WT, wild-type Col-O. Data represent mean response from a minimum of 72 seedlings from at least two independent replicate experiments. Error bars represent SE. Asterisks denote responses significantly different from that observed in nph4 controls (no NOA exposure) by Student's t-test (*, 0.05 > P > 0.01; **, P < 0.001). Molecular Plant 2008 1, 129-144DOI: (10.1093/mp/ssm013) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 5 Root Gravitropism and Auxin Sensitivity in aux1 Missense Alleles. (A) Root gravitropic re-orientation response of wild-type Col-O (WT) and aux1 mutant seedlings. Seedlings were grown on vertically oriented plates for 3 d in darkness, followed by 90° rotation of the plate to induce change in root growth orientation (graviresponse). After 24 h, growth in darkness, root orientations were determined relative to the seedling axis upon re-orientation (see insert, lower right). Although not all aux1 alleles are carried in the Col-O background (see Methods), only the Col-O response is shown, as other wild-types responded similarly (data not shown). Data are projected on a wheel diagram that reflects root orientation at the end of the experiment, where 0° is no change in growth direction after re-orientation, 90° is complete downward re-orientation (note nearly perfect downward re-orientation of WT roots), 180° is growth in the polar opposite horizontal direction, and 270° is upward growth. Data represent percent of total seedlings in each response range from a minimum of 63 seedlings from at least two replicate experiments. (B) Root elongation response of wild-type Col-O (WT) and aux1 missense alleles after exposure to IAA. Seedlings were grown on vertically oriented plates for 3 d in white light, then transferred to new vertically oriented plates containing the indicated concentration of IAA and the position of the root tip for each seedling was noted on the plates. Seedlings were allowed to grow for an additional 3 d, after which total root elongation was determined. Data represent the mean response (presented as percent of controls; no auxin treatment) from a minimum of 70 seedlings from at least two replicate experiments. Error bars represent SE. Molecular Plant 2008 1, 129-144DOI: (10.1093/mp/ssm013) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 6 Hypocotyl Phototropism in Wild-Type, nph4 and nph4aux1-201 Seedlings Grown on Auxin. Seedlings were grown on medium containing the indicated concentration of auxin (IAA or NAA) for 3 d in darkness and then exposed to 4-h unidirectional BL (0.1 mmol m−2 s−1) to induce phototropic curvature. Data represent the mean responses from a minimum of 40 seedlings from at least two replicate experiments. Error bars represent SE. Asterisks denote responses significantly different from that observed in nph4 controls (no auxin exposure) by Student's t-test (*, 0.05 > P > 0.01; **, P < 0.001). Molecular Plant 2008 1, 129-144DOI: (10.1093/mp/ssm013) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 7 Total Auxin Content and AUX1 Promoter Activity in Hypocotyls of Phototropically Stimulated Seedlings. (A) Total free IAA contents in hypocotyls of wild-type Col-O (WT), nph4 nph4aux1-201 and phyA seedlings exposed to phototropic stimuli. IAA contents were determined by gas chromatography–selected reaction monitoring–mass spectroscopy (GC–SRM–MS) as described in the Methods. Data represent the mean IAA content from three independent biological samples. Error bars represent standard deviation. (B) β-glucuronidase (GUS) activity staining in etiolated AUX1Promoter::uidA seedlings exposed to different light conditions. Seedlings were grown in darkness for 3 d, then placed in unidirectional BL (0.1 mmol m−2 s−1) (middle panel; BL), unidirectional BL supplemented with RL (1.6 mmol m−2 s−1) from above (bottom panel; BL+RL), or kept in darkness (top panels, dark) for an additional 4 h, followed by staining for GUS activity (see Methods). Left portion of figure shows GUS staining in the hypocotyl:root junction, while insert shows staining in the root tip (positive control); right portion of figure shows staining in the elongation zone of the hypocotyl. (C) β-glucuronidase (GUS) activity in hypocotyl extracts from etiolated AUX1Promoter::uidA seedlings exposed to different light conditions. Seedlings were grown in darkness for 3 d, then placed in unidirectional BL (0.1 mmol m−2 s−1; BL), unidirectional BL supplemented with RL (1.6 mmol m−2 s−1; BL+RL), or kept in darkness (dark) for an additional 4 h, followed by tissue collection and fluorimetric GUS activity assay (see Methods). Data represent the mean response of two biological replicates assayed over four incubation times. Error bars represent SE. Molecular Plant 2008 1, 129-144DOI: (10.1093/mp/ssm013) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions