Control 50 ng/l 250 ng/l 500 ng/l (A) IAA in 3-mm tip (B) IAA diffused into agar Time after labeled Trp application (min) Labeled IAA (%) IAA (pg tip -1 ) 100 200 300 400 500 0 0 20 40 60 80 0 30 60 90 120 Time after labeled Trp application (min) IAA (pg tip -1 30 min -1 ) 0 100 200 300 400 500 0 20 40 60 80 0-30 30-6060-9090-120 Labeled IAA (%) [ 13 C 11 15 N 2 ]-Trp concentration Supplemental Figure 1. IAA levels and incorporation rate of stable isotope label into IAA in tip, and diffusible IAA after [ 13 C 11 15 N 2 ]-Trp application to top surface of 3-mm coleoptile tip sections. [ 13 C 11 15 N 2 ]-Trp at indicated concentrations was applied to surface of coleoptile (details in Supplemental Figure S2). After incubation for given time periods, IAA was quantified by GC-SIM-MS and proportion of IAA labeled with 13 C 15 N was calculated. (A) Amount of free IAA in section (top), and proportion of IAA labeled with [ 13 C 9 15 N 1 ] (bottom). (B) Amount of diffusible IAA (top), and proportion of labeled IAA (bottom). Supplemental Figure S1
Transcript
Control
50 ng/l
250 ng/l
500 ng/l
(A) IAA in 3-mm tip (B) IAA diffused into agar
Time after labeled Trp application (min)
La
be
led
IA
A (
%)
IA
A
(pg
tip
-1)
100200300
400500
0
0
20
40
60
80
0 30 60 90 120
Time after labeled Trp application (min)
IA
A
(pg
tip
-1 3
0 m
in-1)
0100
200300400500
0
20
40
60
80
0-30 30-60 60-90 90-120
La
be
led
IA
A (
%)
[13C1115N2]-Trp
concentration
Supplemental Figure 1. IAA levels and incorporation rate of stable isotope label into IAA in tip, and diffusible IAA after [13C11
15N2]-Trp application to top surface of 3-mm coleoptile tip sections.[13C11
15N2]-Trp at indicated concentrations was applied to surface of coleoptile (details in Supplemental Figure S2). After incubation for given time periods, IAA was quantified by GC-SIM-MS and proportion of IAA labeled with 13C 15N was calculated.(A) Amount of free IAA in section (top), and proportion of IAA labeled with [13C9
15N1] (bottom).(B) Amount of diffusible IAA (top), and proportion of labeled IAA (bottom).
Supplemental Figure 2. Maize coleoptile (20-mm segment) was detached from seedlings (primary leaves were removed), and labeled Trp was applied locally as indicated by red staining (0.01% neutral red solution). (1) Labeled Trp solution was added to a silicon tube capped onto top surface of coleoptiles.(2) – (5) Labeled Trp was applied to the inner region of the coleoptile using capillary tube.
Supplemental Figure S2
Supplemental Figure 3. IAA labeling method for Figure 1.13C11
15N2-Trp-containing agar block was placed onto 0-1 mm top surface of intact maize coleoptile. After 15 min feeding, the agar was replaced by a cold Trp-containing agar block and incubation was continued for 0, 30, 60, or 90 min.
Supplemental Figure S3
(A) (B)
Supplemental Figure 4. NPA and BFA treatment method for Figure 4 (A), and Figures 5, 6, and 7 (B).(A) Maize coleoptile segment (3-mm) was excised, then 10 mM KPB, pH 6.7, NPA and BFA in KPB were applied using a capillary tube to the inner 2-mm region as indicated by red staining (0.01% neutral red solution). Then, coleoptile tips were placed on an agar block.(B) A 20-mm segment of maize coleoptile was detached and primary leaves were removed. A capillary tube was used to apply 10 mM KPB, pH 6.7, NPA and BFA in KPB to the inner top region of the coleoptile as indicated by red staining. Coleoptile segments were positioned on agar plate.
Supplemental Figure S4
Rat
e o
f cu
rvat
ure
00.020.040.060.080.1
0.120.140.160.18
a b c d
(B)
yx
vascular system(A)
a b c d
g
Thinner side (where leaves emerge)
Supplemental Figure 5. (A) Coleoptile segments (2-cm long) were placed in vertical direction with four different
coleoptile positions indicated in a, b, c, d. Photos were taken after 3 h of gravi-stimulus (bottom).
(B) (B)Rates of curvature in each group in (A) were calculated by x/y as indicated below. Data were obtained from 10 segments for each experiment and values are means ±SE.
Supplemental Figure S5
0
60
120
180(min)
Mock
PEO-IAA
2 20 200 (M)
Supplemental Figure S6
Supplemental Figure 6. Effect of PEO-IAA on gravitropic curvature using the abrasion method for PEO-IAA treatment. A 20-mm coleoptile section was abraded with fine aluminum powder as described by Mori et al. (2005). PEO-IAA at indicated concentrations was applied to inside of 0 to15-mm regions of coleoptiles, and coleoptile sections incubated with PEO-IAA solutions for 30 min. Bases of coleoptiles were fixed in agar, then coleoptiles were tilted horizontally.
Control
NPA 20 M
BFA 25 M
TIBA 20 M
0 60 180 (min)
144 158 162
418
266
500
401
382
514
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Inhibitors
Supplemental Table S1
Supplemental Table 1. Effect of IAA transport inhibitors on IAA accumulation at coleoptile tips
Coleoptiles were abraded and then incubated with inhibitor solutions. After indicated times, the top 2-mm sections of coleoptiles were harvested for analysis. Values are averages of two independent experiments.
