1. Volume 9, Issue 1, Pages 101-112 (January 2016)
Clathrin-Mediated Auxin Efflux and Maxima Regulate Hypocotyl Hook Formation and Light-Stimulated Hook Opening in Arabidopsis 
Qinqin Yu, Ying Zhang, Juan Wang, Xu Yan, Chao Wang, Jian Xu, Jianwei Pan 
Molecular Plant 
Volume 9, Issue 1, Pages (January 2016)
DOI: /j.molp
Copyright © 2016 The Author Terms and Conditions

2. Figure 1
Time-Course Analyses of Hook Development and Auxin Maxima in Clathrin-Deficient Mutants.
(A–C) Dynamic changes of hook curvature in the wild-type and clc2-1 clc3-1 seedlings.
(D–F) Time-course analysis of DR5:GFP expression pattern in the hook region of the wild-type and clc2-1 clc3-1 seedlings.
(C) The mean angles of hook curvature at the different development stages.
(F) The relative intensity of the DR5:GFP signals at the concave side of the hooks. Hook curvature and GFP signals in the etiolated seedlings were examined at different time points (72, 96, 120, and 144 h after sowing; as indicated at the top of the panels).
Data shown are means ± SD. Double and triple asterisks indicate P < and , respectively (Student's t-test, compared with the wild-type). Scale bars represent 500 μm (A and B) and 200 μm (D and E). n, number of examined seedlings as indicated in (C and F).
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

3. Figure 2
Reduced Effects of Auxin Overproduction on Hook Formation in Clathrin-Deficient Mutants.
(A–C) Time-course analysis of hook curvature in YUC1-OE and YUC1-OE clc2 clc3 seedlings. (A) The mean angles of hook curvature at the different development stages (n = 37–90 seedlings each). (B and C) Representative images of hook at 96 and 144 h after sowing.
(D–H) Analysis of distribution of DR5:GFP signals at the concave and convex sides of the hooks in YUC1-OE and YUC1-OE clc2 clc3 seedlings. (D and E) At 96 h after sowing; (F and G) at 144 h after sowing. Panels on the right are magnifications of the dotted boxes in panels on the left. (H) The average ratio of GFP signals between concave and convex sides (n = 21–42 seedlings each; D–G).
Data shown are means ± SD. Single, double, and triple asterisks indicate P < 0.05, 0.01 (A) and (H), and , respectively (Student's t-test, compared with the wild-type or YUC1-OE lines). Scale bars represent 500 μm (B and C) and 100 μm (D–G).
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

4. Figure 3 Effects of Auxin Transport Inhibitors on Hook Formation.
(A–K) Representative images of hook curvature in the presence of auxin transport inhibitors. The wild-type and clc2-1 clc3-1 seeds were vertically germinated for 96 h on 0.5× MS agar plates supplemented with mock (DMSO; A and B), 1-NOA (30 μM; C–E), NPA (5 μM; F–H), and TIBA (1 μM; I–K), respectively. (E, H, and K) Quantification analysis of the inhibitory effects of auxin transport inhibitors on hook curvature (E, n = 101–145 seedlings each; H, n = 70–75 seedlings each; K, n = 101–166 seedlings each). The inhibitory effects of auxin transport inhibitors on hook curvature in the wild-type and clc2-1 clc3-1 are presented as the relative inhibition rates (%) relative to the corresponding mock control (A and B), respectively.
Data shown are means ± SD. Triple asterisks indicate P < (Student's t-test). Scale bars, 500 μm.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

5. Figure 4
Effects of Auxin Transport Inhibitors on Auxin Maxima during Hook Formation.
(A–K) The expression pattern of DR5:GFP in the hook region of 96-h-old wild-type and clc2-1 clc3-1 seedlings treated with mock (DMSO), 1-NOA (30 μM; C–E), NPA (5 μM; F–H), and TIBA (1 μM; I–K), respectively. (E, H, and K) The relative intensity of the GFP signals at the concave side (E, n = 24–38 seedlings each; H, n = 38–78 seedlings each; K, n = 44–80 seedlings each).
Panels on the right are magnifications of the dotted boxes in panels on the left. Single, double, and triple asterisks indicate P < 0.05, 0.001, and , respectively (Student's t-test, compared with the corresponding mock control). Scale bars, 100 μm.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

6. Figure 5
Reduced BL-Triggered Hook Opening in Clathrin-Deficient Mutants.
(A–G) Hook curvature in 96-h-old wild-type and clc2-1 clc3-1 seedlings upon BL illumination. (G) Mean angles of hook curvature in the wild-type and clc2-1 clc3-1 (n = 110–151 seedlings each). Different time lengths (0, 3, and 6 h) of exposure to BL (2 μmol m−2 s−1) are indicated at the top of the panels.
Arrows indicate BL direction. Data shown are means ± SD. Single and triple asterisks indicate P < 0.05 and , respectively (Student's t-test, compared with the corresponding wild-type control). Scale bars, 1 mm.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

7. Figure 6 Kinetic Effects of BL on Auxin Maxima in the Hook.
(A–G) The expression pattern of DR5:GFP at the concave side of the hooks in 96-h-old wild-type and clc2-1 clc3-1 seedlings. (G) The relative intensity of the GFP signals at the concave side of the hooks (n = 30 seedlings each). Treatment conditions are the same as in Figure 5.
Solid and dashed arrows indicate BL and auxin flow direction, respectively. Data shown are means ± SD. Triple asterisks indicate P < (Student's t-test, compared with the corresponding wild-type control). Scale bars, 200 μm.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

8. Figure 7
Subcellular Localization of PIN3 during Hook Formation and BL-Triggered Hook Opening.
(A–D) PIN3-GFP localization in the hook region in 96-h-old wild-type and clc2-1 clc3-1 seedlings in darkness or illuminated with BL (2 μmol m−2 s−1) for 3 h. Left panels, cortex view showing cortical cells. Right panels, middle view showing epidermal and cortical cells at the concave and convex sides.
(E and F) Average ratio of the lateral to basal GFP intensities in cortical cells in the middle view (n = 20 cells from five hooks each).
(G) The relative intensity of PIN3-GFP at the basal PM of epidermal cells of the concave side in the middle view (n = 20 cells from five hooks each).
Arrows and arrowheads show BL direction and PIN3-GFP localization, respectively. Ep, epidermal cells; Co, cortical cells. Data shown are means ± SD. Single, double, and triple asterisks indicate P < 0.05, 0.001, and (Student's t-test), respectively. Scale bars, 50 μm.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

9. Figure 8 Endocytosis of PIN3-GFP in Cortical Cells of the Hook.
(A and C) Treatments with CHX (50 μM) for 120 min in the wild-type and clc2-1 clc3-1.
(B and D) Treatments with CHX plus BFA (100 μM) for 120 min in the wild-type and clc2-1 clc3-1.
(E) The average number of PIN3-GFP-labeled BFA bodies in the wild-type (n = 1901 cells from 73 hooks) and clc2-1 clc3-1 (n = 2301 cells from 77 hooks).
(F) The relative size of PIN3-GFP-labeled BFA bodies in the wild-type (n = 375 BFA bodies from 22 hooks) and clc2-1 clc3-1 (n = 296 BFA bodies from 27 hooks).
Arrows and arrowheads show BL direction and PIN3-GFP-labeled BFA bodies, respectively. Data shown are means ± SD. Triple asterisks indicate P < (Student's t-test). Scale bars, 30 μm.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

10. Figure 9
Simplified Action Modes of Clathrin on Auxin Maxima and Hook Development.
(A) Auxin maxima formation in the 96-h stage hook of the wild-type and clc2-1 clc3-1. Auxin maxima are visualized by the DR5:GFP signals. Red arrows and their size indicate directions and amounts of auxin flow, respectively.
(B) Schematic diagram summarizes the impact of clathrin, ethylene, and light on auxin maxima during hook formation and opening from this work and previous reports (Vandenbussche et al., 2010; Wu et al., 2010; Zádníková et al., 2010). Arrows and T-bars show positive and negative effects of auxin maxima formation or hook formation and opening, respectively. AUX1/LAX3, AUXIN RESISTANT 1/LIKE AUX1 (LAX) 3; PIN3, PIN-FORMED 3; TAA1/TAR2, TRYPTOPHAN AMINOTRANSFERASE 1/TAA RELATED 2; ABCB19, ATP-binding cassette (ABC) transporter B19. Question marks denote hypothetical signaling or interactions for which evidence is currently lacking.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions