Supporting Information legends
Figure S1. Derivatization of IPyA and IAAld by cysteamine.
(a) MS/MS spectrum of IAAld-TAZ after derivatization of IAAld by cysteamine solution.
The product ion scan was measured under optimized MS conditions (see Experimental
procedures) with a collision energy of 15 eV. (b) MS/MS spectrum of IPyA-TAZ after
derivatization of IPyA by cysteamine solution. The product ion scan was measured under
optimized MS conditions with a collision energy of 20 eV. (c) The pH effect on derivatization
recovery of IAAld (black squares) and IPyA (white squares) was tested with 0.25 M
cysteamine solutions at pH 4.0 – 10.0 (adjusted by 1M HCl or 25% NH4OH). Before LCMS/MS analysis, 10 pmol of both analytes were incubated for 1 hour and evaporated to
dryness. The optimal pH for the derivatization reaction was found to be pH 8.0. (d) Improving
the sensitivity of IPyA determination after derivatization with cysteamine compared to
derivatization using hydroxylamine according to the previously published method (Tam and
Normanly, 1998). 20 mg of Arabidopsis seedling material (Col-0) was extracted, halved
before derivatization and purified by the SPE method described in Experimental procedures.
After derivatization, the oxime forms of IPyA and 2H4-IPyA were detected using the MRM
transitions 219.2 > 130.1 and 223.2 > 133.1, respectively, and the thiazolidine forms were
measured under optimized MS conditions (see Table 2). (e) Representative chromatograms of
Arabidopsis extracts derivatized by 0.25 M solution of cysteamine (pH 8.0) under optimized
conditions. Stable isotope-labelled internal standards, 10 pmol of [2H4]-IPyA and [13C6]IAAld, were added during extraction of 10 mg Arabidopsis seedlings (Col-0) and the extracts
were purified by the protocol described in Experimental procedures.
References
Tam, Y.Y. and Normanly, J. (1998) Determination of indole-3-pyruvic acid levels in
Arabidopsis thaliana by gas chromatography–selected ion monitoring-mass spectrometry. J.
Chrom. A, 800, 101-108.
Figure S2. Test of buffer extraction capacity and plant matrix effects on IAA recovery.
(a) Test of extraction capacity of the sodium-phosphate buffer. Different amounts of plant
tissue was extracted and purified on C8 (black squares) and HLB (white squares) SPE
columns. The extraction procedure is linear up to 100 mg of plant material and independent
on the stationary phase of the SPE columns. (b) Test of the plant matrix effect on the recovery
of the [13C6]-IAA internal standard added after purification of different amounts of plant
tissue on silica based (C8, black squares, solid line) and polymer based (Oasis HLB, white
squares, dotted line) solid phase extraction columns.
Figure S3. Test of different loading conditions on the recovery of IAA metabolites after
purification by HLB solid phase extraction.
Different loading conditions (pH 2.7, black bars; pH 7.0, white bars) were used for
purification of IAA metabolites on HLB columns, and the recovery was calculated.
Figure S4. Test of different extraction and purification protocols for IAA and IBA
quantification and IBA spiking experiments of Arabidopsis extracts.
(a-d). Comparison of three different extraction methods and three solid phase extraction
protocols for the recovery of IAA and IBA. Comparison of recoveries from three different
extraction buffers (80% methanol, white bars; 65% isopropanol/0.2M-imidazole buffer (pH
7.0), grey bars; sodium-phosphate buffer (pH 7.0), black bars) and three SPE protocols (C8,
C18 and MAX columns) for purification of IAA (a,c) and IBA (b,d). Samples (10 pmol of
IAA and IBA in mixture) were purified without (a,b) and with plant matrix (20 mg of plant
material) (c,d).
(e-f). Different concentrations of IBA (0, 0.1, 1 and 10 pmol) were added to Arabidopsis
extracts (20 mg fresh weight), extracted with 50 mM Na-phosphate buffer (pH 7.0) containing
0.025% DEDTCA and purified by the mixed-mode anion exchange sorbent. To some of the
extracts, 1 pmol [13C8,15N1]-IBA internal standard was also added.
Figure S5. Representative MRM chromatograms of 20 mg samples from wild-type (Col-0; a)
and mutant (sur1-3; b) plants.
Figure S6. Multivariate modelling of IAA metabolite profiles.
Principal component analysis plots show the correlation between observations (samples, black
dots) with the variables (metabolites, red squares). Components are 1 and 2, which in each
plot contain 60-90 % of the total variation of the data. The closer the dots are to the variables,
the stronger the correlation. (a) Mutant line shoots versus mutant line roots – 35S:YUC1/sur13/sur2-1(R/S); (b) Col-0 versus two ecotypes (Ler, Ws), shoots (S); (c) Col-0 versus two
ecotypes (Ler, Ws), roots (R).
Figure S7. IAA metabolite profiles in the estradiol inducible lines pMDC7:TAA1 (TAA1ox),
pMDC7:YUC6 (YUC6ox) and the empty vector wild-type control pER8.
IAA, the IAA precursors (ANT, TRP, TRA, IAOx, IAM, IPyA, IAN, IAAld), conjugates
(IAAsp and IAGlu) and the catabolite oxIAA were quantified in root (white bars) and shoot
(black bars) tissues. Samples were analyzed in four independent biological replicates, and
error bars represent the SD. Asterisks indicate statistically significant difference between Col0 and other ecotypes and mutant lines (shoot and root tissues) at 95 % confidence level in a
one-way ANOVA analysis.
Table S1. Auxin metabolite profiling of Arabidopsis wildtype and mutant lines.
Levels of IAA metabolites in 10 DAG Arabidopsis seedlings of the ecotypes Col-0, Ws and
Ler, the mutant lines 35S:YUC1, sur1-3 and sur2-1 and the estradiol inducible lines
pMDC7:TAA1 And pMDC7:YUC6 (pER8 is the empty vector wildtype control). 20mg FW
samples of root and shoot tissues were extracted in sodium phosphate buffer, partially
derivatized by cysteamine, purified by one step SPE and quantified using LC-MRM-MS.
IAEt, IBA, IAAla, IAGly, IALeu, IAPhe, IATrp and IAVal were not detected.
Method S1. Preparation of stable isotope-labelled internal standards.
(a) Synthesis of [indole-2H4]-IPyA and (b) synthesis of [indole-2H5]-IAOx.