Supporting Figure legends
Figure S1. Expression and purification of AtYUC1-His protein analyzed by SDS-PAGE.
(a) CBB staining. Whole cell lysate of E. coli harboring the pET3c vector without or with
induction (lanes 1, 2), eluted fraction of the His-tag column of pET3c (lane 3), whole cell
lysate of E. coli harboring the AtYUC1-His-expression plasmid without or with
induction (lanes 4, 5), and eluted fraction of His-tag column of AtYUC1-His (lane 6).
About 2-8 g/lane of protein was loaded. (b) Western-blot analysis with anti-6-His
antibody to detect recombinant AtYUC1-His protein. Ten-time diluted samples were
used for analysis. (c) Western-blot analysis with anti-AtYUC1. Samples were loaded as
in b.
Figure S2. Enzymatic properties of recombinant AtYUC1-His proteins.
(a) Rate of IAA production from IPyA correlated with enzyme amount. At least up to 30
g of partially-purified AtYUC1-His protein, activity increased linearly. (b) Amount of
IAA production from IPyA correlated with enzymatic reaction time. Reaction solution
contained 25 g of partially-purified AtYUC1-His protein. At least up to 50 min, activity
increased linearly. (c) Full mass spectra of authentic IAA (IAA-TMS2; above) and
reaction product (below). Authentic IAA and reaction product were trimethylsilylated
with N-methyl-N-trimethylsilyltrifluoroacetamide. Then, samples were analyzed by
GC-Full-MS. (d) Confirmation of the amount of IAA existed in the IAA peak detected
using HPLC-fluorescence detector. Equal amount of
13
C-IAA to the product IAA
calculated according to the peak area was added in IAA fraction collected from HPLC
eluate. The trimethylsilylated samples were analyzed by GC-SIM-MS. Relative
abundance of obtained signals in
13
C-IAA were almost the same to those of
12
C-IAA
originated form enzyme reaction, showing that the peak of IAA observed in HPLC
analysis is reliable for quantification of IAA in enzymatic assay. Arrow in the
gas-chromatogram indicates peak of IAA.
Figure S3. Effects of methimazole and S-methyl yucasin on AtYUC1-His activity and
molecular docking study of yucasin, Trp and kyn on TAA1.
(a) Effect of S-methyl yucasin and methimazole on AtYUC1-His activity. (b) Chemical
structure of S-methyl yucasin. (c) The crystal structure of TAA1 and the predicted
binding pose and affinity of yucasin in active site of TAA1. The structural data of
TAA1 (PDB ID: 3BWN) were edited by Discovery Studio visualizer 3.0 (Accelrys) and
then converted to PDBQT file by MGL tools (The Scripps research institute). The
ligands (Trp, kyn and yucasin) were docked into TAA1 binding site and binding affinity
was evaluated by Autodock Vina software (Trott and Olson, 2010). The binding pose
with highest affinity score were shown. Trp, substrate and kyn, inhibitor displayed
almost same binding pose and similar affinity scores (estimated log Kd -6.1 and -5.9).
The binding pose of yucasin is different and showed ten-times lower affinity score (-4.9).
(d) Predicted binding affinity (- log Kd
) of inactive control compounds,
estimated
4-chlorobenzoic acid, alanine, methimazole, derivative 2, glycine, S-methyl yucasin
(inactive analog of yucasin) to TAA1.
Figure S4. Effects of yucasin and kyn on maize and Arabidopsis Trp aminotransferase
activity.
(a) Typical HPLC profile to detect IPyA, reaction product from Trp by maize Trp
aminotransferase (AT). Detail procedure is described previously (Koshiba et al, 1993).
Crude enzyme fraction (30-60% ammonium sulfate fraction) was prepared from the
extracts of 0-5 mm coleoptile tips and Trp AT activity was assayed with 100 μl reaction
mixture containing 0.1 M borate-EDTA (pH 8.5), 5 mM Trp, 0.2 mM PLP, and 0.5 mM
-KG. The reaction was performed for 55°C for 30 min, then 100 μl of 100% methanol
was added and after centrifugation an aliquot was analyzed to HPLC with 10-50%
methanol containing 0.2% acetic acid, linear gradient for 40 min at 1 ml/min flow rate.
Retention time of Trp and IPyA are 7.7 and 25.0 min, respectively (arrows). In absence
of -KG, IPyA could not be detected. (b) Full mass spectra of trimethylsilylated
authentic IPyA (IPyA-TMS3) and the eluted product with a retention time at 12.6 min
on gas-chromatography. Peaks of m/z 260, 314, 348 (base peak), 404 and 419 (M+) were
detected as major ion fragments. (c) Effects of yucasin and kyn on Trp AT activity of
maize coleoptiles and Arabidopsis etiolated seedlings. Arabidopsis Trp AT was
extracted from 6-day-old dark grown seedlings and 30-60% ammonium sulfate fraction
was used as the enzyme solution. The activity was assayed with 50 μl reaction mixture
as same as (a), except using 2 mM Trp, with and without yucasin or kyn. After the
reaction for 30 min, the amount of IPyA was determined from the peak area at retention
time 25.0 min. The values obtained from several experiments were normalized and
indicated as relative values (%) against each complete enzyme reaction (100%). The
values are average ±SE (n=3).
Figure S5. Effects of yucasin on high-auxin phenotype induced by the excess exogenous
auxin.
Col-0 and 35S::YUC1 seedlings were grown for 5 days on liquid MS medium containing
20 M yucasin, 50 M L-AOPP or 50 M 5-methyl Trp (Trp analog, 5-mT) with or
without IAA, IAM or Trp. Yucasin did not suppress the phenotype induced by
exogenously-applied excess IAA and IAM. In contrast, non-specific IAA biosynthetic
inhibitor, such as L-AOPP and 5-mT, suppressed high auxin phenotype because of their
toxicity.
Figure S6. Effect of yucasin on DR5::GUS expression in Arabidopsis roots, and on
phenotype of auxin-high production mutant sur2.
(a) Effect of yucasin and PEO-IAA on expression of DR5::GUS in Arabidopsis roots. For
the DR5::GUS reporter assay, seedlings were grown on vertical agar plates for 5 d then
incubated in 10 mM KPB containing a specified concentration of yucasin and PEO-IAA,
a TIR1/AFB auxin receptor antagonist (Nishimura et al., 2009; Hayashi et al., 2012),
with or without 1 M IAA for 7 h. Bar = 2 mm. (b) Effects of yucasin on sur2 seedlings
grown for 5 days in the presence of 50 M yucasin.
Figure S7. Effect of methimazole on IAA level in maize coleoptile tip.
Yucasin and methimazole were applied to the insides of 2-mm coleoptile tips, and then
IAA levels in coleoptile tips were determined by GC-MS. Values are averages of two
experiments.
Figure S8. Estimation for the binding affinity of yucasin on Schizosaccharomyces pombe
flavin-containing monooxygenase (FMO).
(a) The conformation of FAD and NADPH or methimazole in the binding site of FMO
crystal data (PDB ID: 2GVC and 2GV8). The yucasin was imposed to fit the coordinates
of thiourea moiety in methimazole (PDB ID: 2GVC) by Swiss PDB viewer. The binding
affinity of ligands are estimated by the DrugScore X software version 0.88 (Neudert and
Klebe, 2011). The affinity score of NADPH, methimazole and yucasin were -98.271,
-27.245 and +363.741, respectively. NADPH showed highest binding affinity in this
calculation. p-Chlorophenyl moiety of yucasin would collide with the surface of binding
site, when the thiourea moiety in yucasin was located near the coordinates of
methimazole in crystal data. (b) The position of sulfur atom of thiourea moiety would be
crucial for the oxidation of methimazole by FAD co-factor in FMO binding site
(Eswaramoorthy et al., 2006).
Figure S9. Phylogenetic analysis of amino acid sequences of YUC proteins.
The tree was inferred using the UPGMA criterion in MEGA v. 4 (Tamura et al., 2007).
Figure S10. Yucasin treatment reduced root apical meristematic activity.
Arabidopsis Col-0 seedlings were grown on vertical agar plates containing yucasin with
or without IAA for 5 days. Root tip structure was visualized using FM4-64 staining.
White dashed lines mark the division zone of the root tips. Bar = 50 m.
Figure S11. List of primers used for the qRT-PCR analysis.